DNA synthesis in prokaryotes: Replication

7 D N A SYNTHESIS IN PROKARYOTES: REPLICATION DOUGLAS W . SMITH

Department of Biology University o f California, San Diego La Jolla, California 92037 CONTENTS I. INTRODUCTION II. DEHNITIONS

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III. REPLICATION

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A. Basic Features 1. The prokaryotic chromosome 2. Initiation and the origin 3. Chain elongation and the replication fork 4. Termination, segregation, and cell division B. Genetics 1. Bacterial mutants: known enzymes 2. Bacterial mutants: temperature-sensitive dna mutants 3. Bacteriophage mutants C. Enzymes and Other Proteins 1. DNA polymerases 2. DNA ligases 3. Deoxyribonucleases (DNases) 4. Denaturation-renaturation proteins 5. Omegaprotein 6. Plasmid DNA-protein complex D. In vitro DNA Synthesis Systems 1. Permeable cell systems 2. Lysed cell systems E. Mechanisms and Models 1. Discontinuous synthesis 2. The rolling circle model 3. The pre-fork synthesis model IV. CONCLUSIONS

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ACKNOWLEDGEMENTS

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REFERENCES

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7 DNA SYNTHESIS

IN PROKARYOTES:

REPLICATION

DOUGLAS W. SMITH

Department of Biology University of California, San Diego La Jolla, California 92037 I. I N T R O D U C T I O N

The last review of DNA synthesis in this series appeared in 1967 (Shooter, 1967). In this area of biology, much has happened in the intervening five years, including a little progress and many changes in concepts and viewpoints, leading to a general state of expectation and promise for the near future. This review will attempt to survey new advances, trends, and ideas in DNA synthesis, including replication, repair, and recombination. Replication is considered here; repair and recombination will be treated in the next volume in this series. Emphasis will be placed on those resul,ts and ideas which continue to influence current thinking. Because of the extensive subject matter, no attempt at a comprehensive coverage will be made. It is hoped that this article will serve as an introduction to, and a summary of, the state of knowledge concerning DNA synthesis as of January 1972. The interested reader may consult the many reviews in each of the subject areas considered here for more detailed summaries. II. D E F I N I T I O N S

The prokaryotic chromosome is, by definition, the DNA molecule which contains all, or nearly all, of the genetic information of the organism. The term "DNA molecule" will also be used to refer to any DNA fragment of the prokaryotic chromosome. A population of such fragments will comprise a heterogeneous collection of DNA molecules. Such fragments arise from mechanical breakage, or shear, of high molecular weight DNA and from DNA endonuclease digestion. DNA products of polymerizing reactions will also be called "DNA molecules". The prokaryotic chromosome performs two major functions in the cell: replication and transcription. DNA replication is defined as the process by which the chromosome is duplicated. This definition implies two general characteristics of DNA replication: (1) it is a process, probably requiring several biochemical reactions; and (2) it is precisely regulated in time and space, with net synthesis of exactly one DNA molecule per generation time and precise segregation of each of the two newly replicated DNA molecules into each of the two progeny cells. When a DNA molecule is replicated, that part of the new molecule which was present in the old molecule is defined as "parental DNA" and that part which is newly synthesized is defined as "progeny, or daughter, DNA". The old molecule will be termed "the parental DNA molecule", and the two resulting new DNA molecules will be termed "progeny, or daughter, DNA molecules", or "daughter chromosomes". 323

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Three fundamental modes of D N A replication have been envisioned (Delbrfick and Stent, 1957), depending on the distribution of parental DNA in the daughter D N A molecules. If one of the two daughter D N A molecules contains only parental DNA, and the other only daughter DNA, then the mode of replication is termed "conservative". If each daughter molecule contains one completely intact strand of parental DNA and one of daughter DNA, replication is termed "semiconservative". If each of the two D N A strands o f the daughter chromosomes contains some parental D N A and some progeny DNA, then replication is termed "dispersive". Replication modes in which some parental D N A is excised and replaced will be termed "non-conservative". These terms refer explicitly to in vivo modes of D N A replication, but will also be used to describe types of DNA synthesis observed in vitro which mimic the in vivo processes. Thus, semiconservative, or replicative, synthesis, or nonconservative synthesis, may be observed in vitro. The term " D N A synthesis" has two different meanings, a limited meaning and a general meaning. In its limited meaning, D N A synthesis is defined to be any reaction in which low molecular weight DNA precursor molecules are covalently joined to a pre-existing D N A molecule, or are covalently joined to form a new D N A molecule. "Net synthesis" is said to have occurred if the amount of product D N A is greater than the amount of substrate DNA. Thus, at least one D N A synthesis reaction must necessarily occur during the process of D N A replication. In its more general meaning, D N A synthesis is defined to be the set of all processes which include at least one DNA synthesis reaction. Thus, D N A synthesis in prokaryotes includes the processes of replication, repair, and recombination. The terms "repair synthesis" and "extensive synthesis" refer to specific D N A synthesis reactions which are catalysed by E. eoli D N A polymerase I. Any D N A synthesis reaction which mimics this "repair synthesis" reaction, or repair reaction, of DNA polymerase I will be called a repair-type reaction. Thus it is possible that the process of D N A replication will include a repair-type synthesis reaction. In addition to its two major functions, D N A molecules are substrates for at least two other intracellular processes: repair and recombination. Since the linear sequence of deoxynucleotide residues in the DNA molecule comprises the genetic information for the organism, maintenance of the integrity of this sequence is essential for propagation of the species. Organisms have evolved biochemical mechanisms for recognition and repair of any damage to their genetic material. These mechanisms are collectively defined to be "repair processes", and the totality of DNA synthesis reactions found in each of these processes is termed "repair replication". Note that repair replication refers to an in vivo process; repair replication may, or may not, include repair synthesis reactions. When a cell contains two nearly identical chromosomes, or parts of chromosomes, these two chromosomes can interact with resultant exchange of genetic information between the two chromosomes. Such events are defined to be "recombination processes". A double-stranded DNA molecule is said to contain a "nick" when a phosphodiester bond in one of the two D N A strands is broken. A double-stranded DNA molecule is said to contain a "gap" when two phosphodiester bonds in one of the two D N A strands are broken, with excision of the deoxynucleotides between the two breaks. Thus, a D N A molecule with a gap is a double-stranded molecule containing a single-stranded region. A double-stranded D N A molecule is said to suffer a "chop" when two opposing, or nearly opposing, phosphodiester bonds, one in each DNA strand, are simultaneously broken. Standard chemical, biochemical, and genetic terminology, concepts, and abbreviations are used throughout. Thus, a gene is considered to be a cistron, and a cis-acting protein

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would be one which acts only on the chromosome containing the structural gene for the protein. Further abbreviations used in this article include the following: ssDNA, singlestranded DNA; dsDNA, double-stranded DNA; RF, replicative form, replication form; RI, replication intermediate; poly d (A-T), alternating copolymer of 5'-deoxyadenylate and 5'-thymidylate residues; poly(dC:dG), homopolymer of poly(5'-deoxycytidylate) and poly(5'-deoxyguanidylate); poly(U,G), random copolymer containing uridylate and guanidylate residues; DNase, deoxyribonuclease; ATPase, adenosinetriphosphatase; dNTP, deoxyribonucleoside-5'-triphosphate containing any base; dNDP, deoxyribonucleoside-5'-diphosphate containing any base; dNMP, deoxyribonucleoside-5'-monophosphate containing any base; BU, bromuracil; dBUTP, deoxybromuridine-5'-triphosphate; AdR, deoxyadenosine; CdR, deoxycytidine; SDS, sodium dodecyl sulfate; DEAE, diethylaminoethyl; pCMB, para(chloro)mercuribenzoate; pHMB, para(hydroxy)mercuribenzoate; NEM, N-ethyl-maleimide; DNP, dinitrophenol; EDTA, ethylenediaminetetraacetate; EGTA, ethylene glycolbis(fl-amino-ethyl-ether)N,N'-tetraacetate; tris,tris(hydroxymethyl)aminomethane; araC, 1-fl-D-arabinofuranosylcytosine; CM, chloramphenicol; PEA, phenethyl alcohol; NNG, N-methyl-N'-nitro-N-nitrosoguanidine; MMS, methylmethane sulfonate; UV, ultraviolet. III. REPLICATION A. Basic Features 1. The prokaryotic chromosome Most prokaryotic chromosomes are a single piece of nucleic acid, usually a single doublestranded DNA molecule. However, they can be single or double-stranded DNA or RNA, in one or a few pieces, circular or linear. The bacterial chromosome is a single long circular DNA molecule, as visualized either by autoradiography (Cairns, 1963a, b) or by electron microscopy (Bode and Morowitz, 1967; MacHattie et aL, 1965), consistent with genetic analysis (Jacob and Wollman, 1961). All mitochondrial DNA species so far examined are double-stranded, and many are closed circular molecules. Many species, isolated from diverse sources, are nearly uniform in size, with a contour length of about 5 microns and a molecular weight close to 107 daltons (Van Bruggen et aL, 1968; Sinclair et ak, 1967). This subject has been recently reviewed (Helinski and Clewell, 1971; Borst and Kroon, 1969; Nass, 1969). The smallest known prokaryotic chromosomes are those of the RNA bacteriophages such as MS2, R17, f2, and Qfl, where the chromosome is a single RNA chain of molecular weight about 106 daltons (for reviews, see Hofschneider and Hausen, 1968; Cold Spring Harbor Symposium, 1969; Calendar, 1970; Stavis and August, 1970). The small polyhedral phages such as ~ × 174 and $13 and the small filamentous phages such as fd and M13 each have a chromosome which is a circular, single-stranded DNA molecule of molecular weight about 2 × 106 daltons (Sinsheimer, 1968; Ray, 1968; Marvin and Hohn, 1969). Larger bacteriophage usually have a single double-stranded DNA molecule as their chromosome, which often circularizes at some stage during the viral life cycle (Thomas, 1966; Thomas et aL, 1968; Fiers and Sinsheimer, 1962). Plasmids, or stable extrachromosomal DNA elements found in prokaryotic systems, including fertility factors (F and I), colicinogenic factors (Col factors), and drug resistance factors (R factors), are further examples of prokaryotic chromosome-like elements. Their DNA is double-stranded and often circular (for reviews, see Falkow et aL, 1967; Meynell et al., 1968; Curtiss, 1969; Campbell, 1969; Novick, 1969; Falkow et al., 1969).

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FIG. 1. Electron micrographs of colicinogenic factor Col El DNA, with examples of form I DNA (closed supercoiled circular DNA), of form II DNA (open circular DNA), and of a circular dimer. Micrograph prepared by a modification of the Kleinschmidt technique (Kleinschmidt et al., 1963) by D. Blair. Two forms of double-stranded circular DNA are found. In form I, both DNA strands are covalently closed circles, and in form II, only one of the two strands is a covalently closed circle (Fig. 1). Initially discovered in polyoma viral D N A (Dulbecco and Vogt, 1963; Weil and Vinograd, 1963) and in phage 4 x 174-infected E. coli (Burton and Sinsheimer, 1963; Kleinschmidt et al., 1963), form I sediments more rapidly than does form II (Burton and Sinsheimer, 1965; Vinograd et al., 1965; Jansz and Pouwels, 1965), can be converted into form II with mild pancreatic DNase digestion (Vinograd et aL, 1965; Roth and Hayashi, 1966), and can be distinguished from form II visually in the electron microscope (Vinograd et el., 1965; Roth and Hayashi, 1966). Form I, a more compact twisted form (Vinograd et aL, 1965; Vinograd and Lebowitz, 1966), will bind intercalative dyes such as ethidium bromide to a lesser extent than will form II (Radloff et al., 1967; Wang et al., 1967, Crawford and Waring, 1967; Bauer and Vinograd, 1968; Bujard, 1968; Ruttenberg et al., 1968), permitting its separation from form II on the basis of buoyant density using combined CsCl-ethidium bromide density gradients (Le Pecq, 1965; Waring, 1966). The physiological role of the twisted circular form I remains unclear; it may simply serve as a resting stage for the genetic element in the cell, or it may be a replication intermediate (for example, see Fig. 23B). Properties of circular D N A have been recently and extensively reviewed by Helinski and Clewell (1971).

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Early st udies with the bacterial chromosome revealed certain features of DNA replication in prokaryotes which continue to be true and must be explained in any detailed understanding of the process (for a general discussion, see Lark, 1969, and Becker and Hurwitz, 1971). Replication is initiated at a nonrandom, heritable region of the chromosome called the origin, and daughter strand synthesis and elongation proceeds sequentially from this origin along the chromosome at one or a few replication points called growing points or replication forks. This process is very precisely regulated, both in time and in net amount of DNA synthesized. Exactly one doubling of the chromosome content occurs per cell per generation time, and cell division is closely coupled to the DNA replication process. These basic features are considered in somewhat more detail below.

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Hanawalt and co-workers (Maaloe and Hanawalt, 1961 ; Hanawalt et aL, 1961), using the phenomenon of thymineless death (Cohen and Barner, 1954), and amino acid and/or uracil starvation indirectly showed that protein and/or RNA synthesis is required to initiate a round of DNA replication. More direct experiments have verified and extended these observations (Lark et aL, 1963; Soska and Lark, 1966; Anraku and Landman, 1968). A protein or RNA factor seems required for initiation. Thus, in the absence of protein synthesis, already begun rounds of replication are completed, but no new rounds are initiated. A general model for temporal regulation of DNA replication, patterned after the successful operon theory for regulation of protein synthesis (Jacob and Monod, 1961), was proposed by Jacob, Brenner, and Cuzin (1963). In this "replicon model", a replicon is defined to be any genetic element which contains all necessary information for control of its replication. Examples of replicons are the bacterial chromosome and the autonomous FI sex factor. Two chromosomal sites are required for the regulation of replication (Fig. 2): a structural gene whose ultimate product (the initiator) diffuses to the second chromosomal site (the replicator) and interacts in a positive manner to effect initiation of a round of replication. The initiator for a given replicon recognizes only the replicator of that replicon; the regulation systems of different replicons are autonomous. A second postulate, to account for the precision of segregation, requires that a replicon be attached to an intracellular site,

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DOUGLAS W. SMITH

in particular, to the bacterial membrane. Initial supporting evidence centered on the observation that a thermolabile F-lac particle continued replication in a cell at the restrictive temperature only if the cell contained a second F' particle, an F-gal episome. The F-gal supposedly provides initiator for the F-lac episome, an episome with the same replicator (Jacob et al., 1963; Cuzin and Jacob, 1967). The putative initiator has yet to be isolated. Inhibitor studies, however, have provided indirect information concerning its existence and nature, although many control experiments are necessary before such experiments can be convincingly interpreted (Cooper and Weusthoff, 1971; Lark, pers. comm. ; Copeland, 1971). Several proteins involved in DNA replication can be synthesized in the presence of a low concentration, e.g., 25/~g/ml, of chloramphenicol (CM), but not in the presence of a high concentration, e.g., 150/~g/ml, of CM; such proteins are sometimes termed "CM-resistant proteins". Examples include proteins induced by phages lambda (Levine and Sinsheimer, 1969c), S13 (Tessman, 1966), M13 (Pratt and Erdahl, 1968), and 4' × 174 (Stone, 1967; Sinsheimer et al., 1962). The 6 × 174 protein, the product of cistron A (Sinsheimer et al., 1967), has been isolated (Levine and Sinsheimer, 1968, 1969b) and must be present continuously for RF synthesis (Levine and Sinsheimer, 1969a). CM-resistant protein synthesis during bacterial membrane (Lark, 1969) and cell wall synthesis (Mandelstam and Rogers, 1958; Lark and Lark, 1960) has also been reported. In E. coli 15, after amino acid starvation to complete rounds of replication, reinitiation will occur in the presence of 25/~g/ml fluorouracil (Lark and Lark, 1964, 1966; Lark and Renger, 1969), suggesting that a CM-resistant protein is required for initiation. Further, thymine starvation of a thymine-requiring E. coil 15 strain results in premature initiation of one of the two progeny DNA chromosomes upon readdition of thymine (Pritchard and Lark, 1964). This premature initiation will not occur in the presence of 25 /~g/ml CM (Lark, 1966), suggesting that a second CM-sensitive protein also is needed for the initiation step. Further suggestive evidence that two substances are needed for initiation of a round of DNA replication is provided by the combined use of two inhibitors, CM and phenethyl alcohol (PEA). PEA, at the correct concentration, appears to be a specific inhibitor of DNA synthesis (Lark and Lark, 1966; Lark, 1966b; Berrah and Konetzka, 1962; Treick and Konetzka, 1964), and to specifically inhibit initiation. After growth in the presence of PEA, several rounds of replication in the presence of 25/zg/ml CM can occur, suggesting that the CM-sensitive substance accumulates in the cells during growth with PEA (Lark and Lark, 1966; Lark et al., 1967). However, PEA appears to prevent synthesis of the CMresistant substance (Lark and Renger, 1969). Since PEA strongly affects membranes (Silver and Wendt, 1967), the CM-resistant substance may be a membrane protein. Further evidence exists that the temporal regulation of initiation depends on the synthesis of particular proteins. Premature initiation results from treatment with nalidixic acid (Boyle et al., 1967), a specific inhibitor of DNA synthesis (Goss et al., 1965) or from thymine starvation (Pritchard and Lark, 1964; Kallenbach and Ma, 1968). This initiation process requires protein synthesis (Pritchard and Lark, 1964; Boyle et al., 1967). Substitution of bromuracil for thymine results in premature initiation in some bacterial strains (Abe and Tomizawa, 1967; Eberle, 1965; Oishi and Sueoka, 1965; Yoshikawa and Haas, 1968; Helmstetter et al., 1968; Lark, 1969). This is probably due to the slower rate of DNA chain elongation effected by bromuracil, with no corresponding change in the time of initiation (Yoshikawa and Haas, 1968; Helmstetter et al., 1968; Lark, 1969). The normal time of initiation could be simply due to the accumulation and attainment of a critical concentration of an initiator substance. In rich media, simultaneous replication at several forks (dicho-

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tomous replication)is observed in E. cob and B. subtilis (Yoshikawa et al., 1964; Oishi etal., 1964; Helmstetter and Cooper, 1968; Bird and Lark, 1968; Ward and Glaser, 1969b). Nevertheless, the rate of DNA chain elongation is independent of the generation time (Helmstetter and Cooper, 1968; Clark, 1968). Thus, the primary regulatory mechanism resulting in the doubling of the chromosome content once per cell per generation time appears to reside in the timing of initiation events, which in turn depends on the overall rate of protein synthesis in the cell. According to the replicon hypothesis, the chromosome transferred during conjugation is part of the F replicon. Since the F factor is found in the donor during transfer, transfer would be dependent on DNA synthesis in the donor and would require initiation of a round of F replicon DNA synthesis. Replication in the donor would leave one daughter chromosome in the donor, and transfer the other to the recipient. Using a thermolabile F-lac episome, Jacob et al. (1963) presented some evidence that replication in the donor, controlled by the F replicon, was required for conjugation. Further studies, involving autoradiography (Herman and Forte, 1964; Gross and Care, 1966) and nalidixic acid-sensitive and nalidixic acid-resistant donors and recipients (Barbour, 1967; Fisher and Fisher, 1968), corroborated these initial findings. However, using thermosensitive mutants for DNA replication (Bonhoeffer and Schaller, 1965) and assaying for recombinant formation (Bonhoeffer, 1966) or enzyme induction (Bonhoeffer et al., 1967), DNA replication appeared necessary only in the recipient. Recent genetic experiments, using these thermosensitive markers in an isogenic background (Stallions and Curtiss, 1971), indicate that DNA replication in the recipient is necessary for recombinant formation, but not for DNA transfer. The earlier experiments, and the need to use isogenic strains, has been recently reviewed and discussed (Curtiss, 1969). Both genetic and physical evidence (Vielmetter et al., 1968a; Cohen et al., 1968; Rupp and Ihler, 1968; Ohki and Tomizawa, 1968; Vielmetter et al., 1968b; Ihler and Rupp, 1969; Vapnek and Rupp, 1970) show that only one of the two DNA strands is transferred from the donor to the recipient during conjugation. For the FI sex factor, the transferred strand is that which forms the denser band in CsCl-poly(U,G) density gradients (Vapnek and Rupp, 1970). In this rather general preparative technique for the isolation of each strand from a bihelical DNA species (Hradecna and Szybalski, 1967), poly(U,G) binds more readily to one of the two F1 DNA strands, resulting in DNA-poly(U,G) complexes of different buoyant density (Fig. 3). Both the transferred strand and that remaining in the donor cell then acquire complementary DNA strands, and the molecules are converted into closed circular forms (Vapnek and Rupp, 1970); DNA replication associated with mating thus appears to occur normally both in the donor and in the recipient. Sucrose gradient and genetic marker analysis of extracts of germinating B. subtilis spores has shown that the origin region of the B. subtilis chromosome in germinating spores is noncovalently attached to some cellular structure (Sueoka and Quinn, 1968; Yamaguchi et al., 1971). Similar experiments in E. cell using DNA radioactively-labeled specifically at the origin and terminus did not lead to similar results (Rosenberg and Cavalieri, 1968); however, recent studies suggest that the origin for initial T4 DNA replication is attached to E. coli cell components (Marsh et al., 1971). To observe the replication origin, the cell population must be synchronized or treated in a way to lead to simultaneous initiation in all cells. Germination of B. subtilis spores (Sueoka, 1966; Wake, 1963), amino acid starvation (Maaloe and Hanawalt, 1961; Anraku and Landman, 1968), membrane selection of E. coli B/r (Helmstetter, 1967) and K12 (Cummings, 1970), dilution and growth of early stationary phase E. cob (Cutler and

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Fraction no FIG. 3. Equilibrium centrifugation in CsCl-poly(U,G) of FI sex-factor DNA strands. Form I supercoiled DNA was isolated usingCsCl-ethidiumbromide equilibriumcentrifugationand converted into form II by introductionof single-strand breaks. Linear ssDNA from form II species was isolated in an alkaline sucrose gradient, mixed appropriately with poly(U,G), and centrifuged for 48 hr at 35,000 rpm at 15°C. The dashed vertical lines represent the peak positions of the complementary strands of aZP-labeled DNA from phage 4II that was added as marker. (a) Sex-factor DNA from unmated donor cells. Two peaks, corresponding to the two complementary DNA strands, are observed. (b) Sex-factor DNA from recipient cells labeled before and during mating. The 3H-label is found only in the denser band, indicating that only this strand is transferred into the recipient during mating, and is then converted into a form I supercoiled DNA molecule. (c) Sex-factor DNA from donor cells labeled before mating and isolated after mating. The 3H-label is found only in the lighter band, indicating that only this strand remains in the donor during mating, and is converted into a form I supercoiled DNA molecule during mating. From Vapnek and Rupp (1970). Evans, 1967), a n d separation of cells by size in sucrose density gradients (Mitchison a n d Vincent, 1965; Bendigkeit et al., 1967) have all been used to o b t a i n synchronized cell populations. Each method has advantages a n d disadvantages (see C a m e r o n a n d Padilla, 1966, for reviews). Using a m i n o acid starvation, Lark a n d co-workers (Lark et al., 1963; Pritchard a n d Lark, 1964) showed that a heritable origin exists in E. coli for at least six generations; such a n origin is also present in S. typhimurium ( C h a n a n d Lark, 1969) a n d in Mycoplasma laidlawii B (Smith a n d Hanawalt, 1968). Sueoka a n d co-workers, using

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FIG. 4. Density distributions of intact DNA molecules from Mycoplasma laidlawii B cells grown for 1.5 hr (0.3 generation) in 5-bromodeoxyuridine medium. Two cultures were prelabeled for 4-5 generations, one with 14C-thymidine (0.15 t~C/ml, 1 t~g/ml), the other with aH-thymidine (0.23 /~C/ml, 1 /zg/ml), and separately transferred into medium containing nonradioactive 5-bromodeoxyuridine (2.4 #g/ml). After growth for 1.5 hr, the cultures were collected, and a typical sheared lysate was prepared from the 1*C-labeled cells. About 109 of the aN-labeled cells were lysed directly on each of two three-layer CsCl gradients, containing half of the 14C-labeled cell lysate in the first layer. The tubes were incubated at 60 ° for 2 hr, then at room temperature overnight. Tube B was briefly manually stirred, and both tubes were centrifuged for 36 hr at 3700 rev/min and 20° in the 40 rotor, followed by drop collection and assay of radioactivity. - - O - - O - - , 14C a c t i v i t y ; - - A - - A - - , aH activity. From Smith (1969).

germinating B. subtilis spores and a combination o f marker frequency comparisons (Yoshikawa and Sueoka, 1963a; Sueoka and Yoshikawa, 1963) and density transfer experiments (Yoshikawa and Sueoka, 1963b; Yoshikawa et al., 1964; Oishi et al., 1964; Sueoka and Yoshikawa, 1965; O'Sullivan and Sueoka, 1967), demonstrated the presence o f a unique origin near the adel6 locus, and a sequential, unidirectional mode o f replication. The D N A itself f o u n d at the origin m a y have unique properties. Yoshikawa (1967,11970b), using density labeling o f germinating spores o f B. subtilis, f o u n d that the daughter D N A synthesized u p o n germination was covalently attached to parental D N A . However, Stein and H a n a w a l t (1972), in similar experiments with E. coli T A U - b a r , synchronized~via amino acid starvation and density-labeled by shifting f r o m bromuracil-containing medium to thymine-containing medium, f o u n d no evidence for such an attachment. A transient attachment, short-lived in the exponentially growing E. coli cells, but longer lived in the germinat-

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DOUGLAS W. SMITH

ing B. subtilis spores, is possible. This is consistent with the pre-fork model of Haskell and Davern (1969) for DNA replication (Fig. 22). However, Stein and Hanawalt (1972) would have detected attachment if such existed longer than about 2 min. Recent evidence using inhibitors suggests that an RNA species is involved in initiation of a round of replication. Genetic experiments had earlier implicated a requirement for transcription of the chromosome origin of phage lambda prior to initiation of lambda DNA replication (Dove et al., 1969, 1971). The antibiotics rifampicin and streptolydigin specifically inhibit the process of transcription (review: Wehrli and Staehelin, 1971); rifampicin inhibits initiation of transcription (Mauro et al., 1969) and streptolydigin inhibits subsequent RNA chain elongation (Schleif, 1969; Cassini et al., 1971). Experiments of K. Lark (1972) with E. coli 15T- using the above two antibiotics, as well as chloramphenicol, indicate that RNA synthesis is required late during replication, following a protein synthesis requirement, for initiation of the next round of replication. This RNA synthesis appears to occur about 10 min prior to initiation, and is also required for the premature initiation observed following thymine starvation (Pritchard and Lark, 1964). This RNA species, apparently stable, can be utilized for subsequent replication cycles. A model of the replication complex, consisting of a complex of proteins self-assembled around an RNA core, was proposed. In vitro incorporation of dNTP's in the cellophane DNA synthesis system (Schaller et al., 1972) is inhibited by pretreatment with EDTA and RNase, but not by pretreatment with RNase alone, further supporting this model (K. Lark, pets. comm.). In similar studies with phage M13 replication, Brutlag, Schekman, and Kornberg (1971) observed that both conversion of the parental single-stranded DNA to the parental RF and replication of this double-stranded form are blocked by rifampicin but not by chloramphenicol. This inhibition was not observed using a rifampicin-resistant E. eoli host. A role for RNA polymerase in initiation of DNA replication was proposed. On the other hand, Silverstein and Billen (1971) found no inhibition by rifampicin of the formation of the parental RF species in q~× 174-infected E. coli. These workers also observed that DNA chain elongation proceeds without concomitant transcription, consistent with earlier observations (see, for example, Donachie and Masters, 1969). Attempts to determine the location of the origin and direction of replication in E. coli F - , F-prime, and Hfr strains have led to contradictory results (Donachie and Masters, 1969). These experiments have utilized (1) marker frequency and gene product analysis in exponentially growing cells (Nagata, 1963a, b; Donachie and Masters, 1966; Vielmetter et al., 1968b); (2) synchronization by amino acid starvation with subsequent phage P1 transduction (Abe and To mizawa, 1967; Espardellier-Joset et al., 1967; Wolf et al., 1968a; Caro and Berg, 1968) or N-methyl-N'-nitro-N-nitrosoguanidine (NNG) mutagenesis (CerdaOImedo et al., 1968; Cerda-Olmedo and Hanawalt, 1968); and (3) synchronization via membrane immobilization (Helmstetter, 1967) with subsequent P1 transduction (Wolf et al., 1968a, b), or N N G mutagenesis (Wolf et al., 1968b; Ward and Glaser, 1969a), or gene dosage effects on enzyme production (Wolf et al., 1968b; Helmstetter, 1968). A better approach has made a direct comparison of marker frequencies between cells grown in rich broth, exhibiting dichotomous replication, and cells grown in glucose-minimal salts medium (Masters, 1970). This avoids uncertainties due to amino acid starvation or use of bromuracil, which is toxic for E. coli (Hackett and Hanawalt, 1966). Most of these results, the latter included, conclude that the replication origin is in the region 40 to 50 min on the E. coli genetic map (see Fig. 7), and that replication proceeds sequentially and unidirectionally in a clockwise manner. This origin is independent of the presence of a lambda prophage on the

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333

E. coli chromosome (Eberle, 1970), as well as the F sex factor. Evidence for bi-directional

replication is presented below. Regarding bacteriophage, the chromosome of phage lambda can be selectively sheared into halves (Radding and Kaiser, 1963), and the two halves isolated in mercury-CszSO, density gradients on the basis of differences in buoyant density (Nandi et al., 1965). Using this technique, Makover (1968a, b) concluded that the replication origin lies in the right half of the lambda chromosome. LePecq and Baldwin (1968), using pulses of bromuracil followed by a thymine chase, reached similar conclusions. Tomizawa and Ogawa (1968) hybridized "replicating" lambda DNA molecules against isolated right and left DNA halves, and found that the earliest replicated part hybridized against the right halves. These results are consistent both with the denaturation mapping studies (Fig. 6) of Schntis and Inman (1970) and with studies of lambda-dv (Fig. 13), a lambda chromosome fragment capable of self-replication (Matsubara and Kaiser, 1968). Simultaneous infection with T4 amber ( a m - ) mutants and "small" defective T4 am* particles yields some am + progeny phage (Mosig, 1970). The yield varies with the map position of the am mutants used, showing a clockwise gradient with a discontinuity between genes 42 and 43. These data are interpreted to mean that replication of the T4 chromosome proceeds unidirectionally clockwise from a unique origin near gene 43. Marker frequency analysis using a T4 transformation system are consistent with this interpretation (Marsh et al., 1971). 3. Chain elongation and the replication f o r k

The critical innovation needed to determine the overall mode of DNA replication was a method for isolation of daughter DNA from parental DNA. E. coli, uniformly density labeled with lSNH4C1, was shifted to "light" ~4NH4Cl-containing medium, grown for varying periods of time, and the DNA isolated and analyzed using centrifugation to equilibrium in CsC1 density gradients (Meselson et al., 1957). This analysis demonstrated that DNA replication proceeds in a semi-conservative manner (Meselson and Stahl, 1958; Rolfe, 1962). Similar analysis of intact replicating bacterial chromosomes in CsC1 gradients (Davern, 1966; Smith, 1969), and the use of other density labels (Lark et al., 1963; Hackett and Hanawalt, 1966), agrees with these conclusions. In addition, the semi-conserved subunits are most probably single DNA strands (Baldwin and Shooter, 1963). Genetic evidence in B. subtilis demonstrating sequential replication was mentioned above. In E. coli, Nagata and Meselson (1968) pulse-labeled the DNA of exponentially growing cultures with all-thymine, permitted growth for varying periods of time, density pulselabeled using laC-glucose and ~SNH4C1 for 0.3 generations, and examined the DNA in CsC1 density gradients. The all-labeled DNA was found transferred to the hybrid density position only at intervals corresponding to multiples of the generation time, demonstrating maintenance of the sequence and direction of replication during growth (see Fig. 5). Other evidence for sequential replication includes the autoradiographic and electron microscopic visualizations of the prokaryotic chromosome, and the B. subtilis marker transfer experiments, mentioned above. Until recently, chain elongation was thought to proceed unidirectionally on the prokaryotic chromosome (Sueoka, 1966; Bonhoeffer and Messer, 1969; Lark, 1969). However, recently Schnrs and Inman (1970), using denaturation mapping by electron microscopy, have shown that the origin of replication in replicating lambda chromosomes (Ogawa etal., 1968) is located about 18~o of the distance from the right end of the lambda vegetative

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map (see Fig. 13), and that replication in the doubly-branched molecules proceeded in both directions in about 70~o of the molecules examined. Figure 6 shows an example of such a molecule examined when partially denatured. Stevens et aL (1971), using hybridization of lambda RNA homologous for specific regions of the lambda chromosome, reached similar conclusions. A similar denaturation mapping study of phage P2 DNA by Schn/Ss and Inman (197 I) showed that, in contrast to the lambda study, most replicating molecules were singly branched, and that replication appeared to proceed unidirectionally. Recent genetic experiments have provided evidence that replication in E. coli also proceeds bidirectionally, in agreement with the suggestive evidence of Yahara (1971). Masters and Broda (1971), using P1 transduction and comparing transductants obtained from cells grown in rich. broth with those obtained from cells grown in glucose-minimal salts medium, concluded that the origin of replication is in the region of the malA and argG loci, at about 60 to 65 min on the E. coli genetic map (see Fig. 7), in fair agreement with earlier findings (see above). Further, replication appears to proceed in both directions, at different rates, to a terminus near the his operon (see Fig. 7). Their results, a compilation from several E. coli strains, are strainindependent, and are independ6nt of the presence and location of the F sex factor on the

D N A S~rHESIS IN PROKARYOTES: REPLICATION

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bacterial chromosome. An example of their findings is shown in Fig. 8. Using P22 transduction of bromuracil pulse-labeled S. typhimurium previously synchronized by amino acid starvation, Nishioka and Eisenstark (1970) had concluded that replication also proceeds bidirectionally in this bacterium. Bird et aL (1971), using deletion mutagenesis effected by insertion of phage mu, also concluded that E. coli replicates in a bidirectional manner, with an origin at about 74 min and a terminus at about 25 min on the E. coil genetic map. The rate of chain elongation has been determined using density labeling techniques (Bonhoeffer and Gierer, 1963; Bird and Lark, 1968), autoradiography (Cairns, 1963a; .

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Lark, 1966b), and pulse labeling of membrane synchronized E. cob B/r cells (Helmstetter and Cooper, 1968). The time C for a round of replication is about 40 rain, and the time D between completion of a round of replication and cell division is about 20 min, for all cell doubling times between 20 min and 60 min (Clark and Maaloe, 1967; Helmstetter and Cooper, 1968; Cooper and Helmstetter, 1968; Helmstetter and Pierucci, 1968; Clark, 1968a, b; Bird and Lark, 1968; Ward and Glaser, 1969b). For a chromosome of molecular weight 2.4 x 109 daltons, the overall nucleotide incorporation would then be about 10s nucleotide pairs per minute. For doubling times between 20 min and 40 min, dichotomous replication necessarily occurs. Dichotomous replication is also observed in "shift-up" experiments, when the growth medium is abruptly changed from a minimal to an enriched medium (Chai and Lark, 1970). For longer growth times, both C and D appear to increase, with C remaining about twice D, in both E. coli B/r (Helmstetter et al., 1968) and in E. coli 15 T - (Lark, 1966; Eberle and Lark, 1967; Chai and Lark, 1970; Bird and Lark, 1970). This subject, and the relation of DNA replication to segregation and cell division, has been

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extensively reviewed (Lark, 1966b; Lark et aL, 1967; Helmstetter, 1969; Lark, 1969). Differences between the above two E. coli strains appear to exist when grown at 21 ° (Urban and Lark, 1971 ; Pierucci, 1972), and recently Kubitschek and Freedman (1971), using nonsynchronized, chemostat grown E. coli B/r, argued that C is 47 min and D is 25 min for all generation times from 20 min to 50 hr. The observed differences in these experiments are possibly due to the different growth conditions. It should also be noted that the chain elongation rate appears to be abnormally low for thymine-auxotrophs of both E. coli and B. subtilis, and to decrease with decreasing concentrations of thymine with no change in the growth rate (Zaritsky, 1970; Pritchard and Zaritsky, 1970; Ephrati-Elizur and Borenstein, 1971 ; Zaritsky and Pritchard, 1971 ; Manor et al., 1971). These observations may be relevant to the interpretation of DNA synthesis experiments using thymine-requiring bacteria at low thymine concentrations. In addition, the probability that replication proceeds bidirectionally necessitates a re-examination of the rate of chain elongation, which may also be different at the two replication forks (Masters and Broda, 1971). Bird and Lark (1970) have determined that the newly replicated small DNA pieces

DNA

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(Okazaki fragments; see below) have the same size, and are synthesized in the same time, at low cell doubling times as at high doubling times. Granted that the time C for a round of replication is in fact longer at low doubling times, this suggests that the overall D N A chain elongation time is regulated by the rate of initiation of synthesis of the small pieces. In view o f the observations of Pritchard and co-workers, it would be interesting to examine the rate of synthesis of the small pieces in a thymine-auxotroph as a function of thymine concentration; such experiments would possibly be relevant to thymineless death (Cohen and Barner, 1954). 1.4

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Ft6.9. Rapid sedimentation of pulse-labeled DNA from gently-lysed Eseherichia coil in neutral sucrose gradients. E. eoli TAU-bar was prelabeled for three generations with. 14C-thymine, washed, and pulse-labeled with 3H-thymidine for 30 sec at 37 °. The concentrated cells were gently lysed with lysozyme-EDTA [see Smith and Hanawalt (1967) for details], briefly mixed, treated with pronase (100/zg/ml, 37 °, 30 min), split three ways and treated further as follows: A. No further treatment (control). B. Treated with deoxycholate (1 ~ , 37 °, 30 min). C. Treated with sarkosyl (0.I ~ , 37 °, 30 min). The treated lysates were centrifuged at 25,000 rpm for 3 hr in 5-20 ~o sucrose density gradients, containing a 6 0 ~ pad; the top or "shelf" of this pad is shown by the vertical dashed line. Sedimentation is from right to left. Fractions were collected, and the DNA acid-precipitated, collected on filters, and assayed for radioactivity. • t4C-thymine prelabel, • 3H-thymidine pulse-label. Figure from Smith (1967). and Y o ung, 1969; Yamaguchi et al., 1971), f r o m E. coli infected with phages ~ × 174 (Knippers and Sinsheimer, 1968a; Salivar and Sinsheimer, 1969), lambda (Salivar and Sinsheimer, 1969; Hallick et al., 1969; Salivar and Gardinier, 1970; K o r n and Thomas, 1971; Kolber and Sly, 1971), T4 (Frankel et al., 1968; A l t m a n and Lerman, 1970a, b; Miller and Buckley, 1970; Miller and Kozinski, 1970a), and M13 (Kluge et al., 1971), and from S. o'phimurium

DNA SYrCrHESlSIN PROKARYOTES"REPLICATION

339

infected with phage P22 (Botstein, 1968; Botstein and Levine, 1968), sediments rapidly in sucrose density gradients. This material can be accumulated on a pad of high concentration sucrose (Smith and Hanawalt, 1967) or CsCI (Knippers and Sinsheimer, 1968a). Treatment with detergents or proteolytic enzymes causes the newly-replicated DNA to sediment with the majority of the DNA fragments, suggesting that the newly replicated DNA is bound noncovalently to cellular material in a high molecular weight, rapidly sedimenting replication "complex". Figure 9 shows typical sucrose gradients of E. coli extracts illustrating these features. Somewhat similar material from B. subtilis has been banded in Renografin gradients (Ivarie and Pen6, 1970). The complex from lambda-infected cells is not found using lambda susN mutants (Hallick et al., 1969; Kolber and Sly, 1971), mutants defective in the regulation of DNA replication (Joyner et al., 1966; Eisen et aL, 1966), but is found using lambda susO and susP mutants (Kolber and Sly, 1971), mutants defective in DNA replication (Brooks, 1965; Joyner et al., 1966). Superinfecting lambda DNA is circularized and apparently becomes associated with the membrane of an immune lysogen, but is not replicated (Salivar and Gardinier, 1970), although some disagreement exists (Korn and Thomas, 1971). DNA synthesizing activity was observed in the rapidly sedimenting material obtained from B. subtilis (Ganesan, 1967, 1968a, b) and from T4-infected E. coli (Frankel et aL, 1968); such material has served as the source of in vitro membrane DNA synthesis systems, discussed below. By varying the multiplicity of infection of E. coli with phages if× 174 or lambda, and employing a membrane assay procedure, Salivar and Sinsheimer (1969) estimated that the E. coli cell contains about 70 possible replication "sites" for each bacteriophage. A membrane-DNA-RNA-protein complex from E. coli, trapped in a Mg + +-sarkosinate layer, has also been studied (Earhart et al., 1968; Tremblay et aL, 1969). Recently, using gentle lysis in 1.0 M NaC1, Stonington and Pettijohn (1971) isolated the E. coli chromosome as a rapidly sedimenting (3200 S) DNA-RNA-protein complex. This complex, of remarkably low viscosity, may be the prokaryotic "nuclear region"; if so, the DNA so obtained probably closely resembles the in vivo DNA as an enzyme substrate. The complex is disrupted by RNase, with a dramatic increase in viscosity, suggesting that the DNA may be held as a compact ball by an RNA network. The DNA found at the growing point has a forked, or branched, structure (Fig. 6,23) as seen visually (Cairns, 1963a; Bode and Morowitz, 1967; Ogawa et al., 1968; Dennis and Wake, 1969). It can be isolated via density labeling (the two daughter arms are of hybrid density, the third arm, containing only parental DNA, has the parental density; the fork is thus of intermediate density), and is particularly fragile and susceptible to shear (Hanawalt and Ray, 1964; Smith and Hanawalt, 1967; Smith, 1969). Further, newly synthesized 17. coli DNA appears to possess some properties of single-stranded DNA (Okazaki et aL, 1968a), as examined using counter-current distribution (Kidson, 1966, 1968), hydroxylapatite binding and nitrocellulose binding (Oishi, 1968a). It also appears to pass through a partially denatured intermediate form (Oishi, 1968b) before becoming native bihelical DNA. In both neutral and alkaline sucrose gradients, the newly replicated DNA has a low molecular weight, of sedimentation coefficient about 10S (Sakabe and Okazaki, 1966; Okazaki et al., 1968a, b; Sadowski et aL, 1968; Oishi, 1968a). These fragments, or short pieces, termed "Okazaki fragments" after their discoverer, are found in all E. coli strains, all phage-infected E. coli, and all B. subtilis strains examined. The fragments are "chased" into higher molecular weight fragments with longer pulse times; an example of such an experiment is shown in Fig. 10. Based on the existence of such fragments, chain elongation mechanisms involving discontinuous modes of synthesis have been proposed (Okazaki et

340

DOUGLAS W. SMITH I

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D N A SYNTHESISIN PROKARYOTES"REPLICATION

34I

of kinetic pulse-labeling experiments performed at low temperature (15 °) and at high cell densities (about l01° cells/ml), Werner (1971a) has provided evidence that thymidine as a pulse-label enters DNA by way of a repair synthesis, whereas thymine as a pulse-label is incorporated during semi-conservative synthesis. He has further proposed that the short pieces observed by Okazaki and others are an artifact of pulse-labeling with thymidine. However, Okazaki et al. (1971) observe no difference in their sucrose gradient profiles (see Fig. 10) when either thymine or thymidine is used during the pulse-labeling. The enzymology of chain elongation is not understood. However, from the basic features mentioned above, it is clear that endonucleases, DNA ligases, and one or more polymerase enzymes are probably involved. In addition, other possibly nonenzymatic protein factors, such as the putative initiator, the renaturation-denaturation protein, and the omega protein may be involved. Some of these possibilities are discussed below (sections III C, E). 4. Termination, segregation, and cell division

This subject has recently been reviewed (Lark, 1969; Helmstetter, 1969). Very little is known about the termination process. The signal for termination of a round of replication is possibly associated with the arrival of the replication fork at the origin, although the possibility of a terminus at a different site of the E. coli chromosome renders this less likely (see Fig. 7; Masters and Broda, 1971; Bird et aL, 1971). Possible unusual structure of the DNA at the terminus or attachment of the terminus to a cell "site", similar to observations of the DNA at the origin, or a termination factor similar to the rho factor (Roberts, 1969), could serve as termination signals in a variety of ways. Termination is defined operationally only in terms of initiation inhibition experiments, e.g., amino acid starvation experiments. No termination-defective conditional-lethal mutants have yet been reported. The daughter chromosomes following a round of replication are precisely segregated into the daughter bacterial cells during the cell division process. E. coli growing in an enriched broth at 37 ° contain eight progeny chromosome arms (Helmstetter and Cooper, 1968); these are found in each of the eight progeny cells three generations later, as determined by tritium autoradiography (Lark, 1966b; Lark et aL, 1967; Lin et aL, 1971). The control mechanisms used to effect this precise segregation pattern are not as yet understood. However, attachment of the chromosome to the cell membrane could automatically lead to segregation during membrane biosynthesis and elongation of the cell; this is a primary idea of the replicon hypothesis (see above), and of a membrane growth model for the regulation of DNA replication (Marvin, 1968). Separation of daughter chromosomes during a round of unidirectional replication can be readily envisioned if replication proceeds asymmetrically (see Lark, 1969, for a general discussion). Alternatively, separation of the daughter chromosomes may occur only after completion of a round of replication and separation of the replicating DNA molecule into two molecules. This is possible with a unidirectional mode of replication by assuming segregation of replicating DNA molecules, i.e., molecules containing one or more replication forks (see Fig. 11A for such an example). With a bidirectional mode of replication, the segregation problem becomes somewhat less difficult to visualize; Fig. 11B shows one possible adaptation of the general model of Fig. 11A to fit a bidirectionally replicating chromosome. The bacterial "nuclear" regions are observed to separate about 30 min before cell division in cells growing with a generation time of about i hr, either by selective staining (see, for example, Murray, 1960) or by phase contrast microscopy (Schaechter et al., 1962). These nuclear regions may be replicating regions of a single chromosome under rapid growth conditions (Chai and Lark, 1970).

342

DOUGLAS W. SMITH

Completion of a round of D N A replication is necessary for cell division to occur (Clark, 1968b; Bird and Lark, 1968; Helmstetter and Pierucci, 1968). As discussed above, cell division occurs a fixed time D after completion of a round of replication in E. coli, although this may not be true for B. subtilis (Donachie et al., 197l). D is about 20 min for cell doubling times between 20 and 60 min, and becomes longer for longer doubling times. Thus, completion of a round of D N A replication appears to "trigger" cell division in E. coll. However, extensive protein synthesis is also required for cell division to occur (Helmstetter, 1969). It has been proposed that cell division occurs when a critical cell mass to D N A ratio is attained (Koch and Schaechter, 1962; Donachie, 1968; Donachie et al., 1968 ; Adler et al.,

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FIG. 11. Possible relationship between DNA replication, segregation, and cell division. The bacterium is assumed to be similar to E. call B/r growing at 37° in a minimal medium. © the replication origin; • the replication terminus (if different from the origin); Y the replication forks, with chain elongation proceeding from the origin to the terminus. The vertical dashed line indicates septum formation at 30 min. The origin may be imagined attached to the cell membrane, and the replication forks and terminus to the cell membrane near the center of growing cell. Figure adapted from Clark (1968b).

1969). Among other biochemical events, a septum is formed generally across the mid-region of the elongated bacterium (Pardee, 1968). Septum formation may be directly coupled to D N A replication (Walker and Pardee, 1968), although probably it is independent of nuclear segregation (review: Ryter, 1968). Genetic analysis is consistent with the above ideas. Mutants of E. call exist in which D N A replication proceeds normally, but which are defective in nuclear segregation and/or cell division (Hirota et al., 1968a, b), indicating that cell division or nuclear segregation does not control D N A replication. Mutants have also been obtained which form septa distal to the segregating nuclear regions, resulting in the production of DNA-less "minicells" in the progeny (Adler et aL, 1967; Hirota et al., 1968a, b). In the case of the Adler mutant, some evidence exists that D N A replication is necessary for the production of the minicells (Clark, 1968b).

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B. Genetics 1. Bacterial mutants: known enzymes (a) DNA polymerase L A major breakthrough in the conceptualization of the DNA replication process was the isolation by DeLucia and Cairns (1969) of the first mutant of E. coil deficient in the Kornberg DNA polymerase (DNA polymerase I). This mutant, obtained from E. cob W3110, is called W3110 polA1, or P3478. Five more pol I- E. coli mutants have since been isolated by DeLucia and Cairns (Gross, 1970, 1972). All five mutants map in the same region of the E. coli chromosome as does polAl (Fig. 7); complementation analysis indicates that all six mutants are in the same gene polA (Gross, 1970, 1972). The polymerase I isolated from E. coli polA6 has altered properties compared with the wild type enzyme (Kelley and Whitfield, 1971). W3110 polA4 produces a cold-sensitive thermolabile polymerase I (Gross et al., 1971). Other polymerase I mutants, in both E. coli (Kato and Kondo, 1970; Zissler and Signer, 1971) and in B. subtilis (Hempstead, 1968) have been obtained, primarily by screening methyl methane sulfonate (MMS) sensitive mutants. Inability of the col factor ColE1 to propagate in W3110 polA1 (Kingsbury and Helinski, 1970) has led to the isolation of two E. coli temperature-sensitive DNA polymerase I mutants, DK214 and DK216 (Kingsbury and Helinski, manuscript in prep.). These apparently map in the polA locus, using reciprocal P1 transduction methods. W3110 polAl is a recessive amber mutant (Gross and Gross, 1969), and the polA locus cotransduces with metE at 20%, located at 75 min on the E. cob genetic map (Fig. 7). W3110 polA1 grows normally in all media and at all temperatures tested (Monk et al., 1971; Kuempel and Veomett, 1970; DeLucia and Cairns, 1969). However, Rosenkranz et al. (1971) observed that W3110 polAl, when grown in a synthetic liquid medium exhibited only a 5~o plating efficiency when plated on nutrient agar plates; we have made similar observations (Ryder and Smith, unpublished results). The low plating efficiency is not observed when the composition of the liquid and solid media are the same. Further, in q~x 174infected W3110 polAl, a greater proportion of RFII molecules are found than in polA + cells, and these appear to contain gaps rather than nicks in the open DNA strand (Schekman et al., 1971). Recombination rates are apparently normal (Gross and Gross, 1969; Kato and Kondo, 1970); however, red- lambda phage (see Fig. 13) grow poorly on W3110 polA1 (Zissler and Signer, 1970, suggesting a possible involvement of DNA polymerase I in some host genetic recombination events. Most episomes propagate normally in W3110 polAl cells. However, the col factor ColEI cannot replicate in W3110 polAl, although it will in polA + revertants, and the col factor ColE2 is unstable in W31 I0 polA1 (Kingsbury and Helinski, 1970). Spontaneous mutation frequencies, both for tonB-trp deletion mutants (Coukell and Yanofsky, 1970) in all six E. coli polA mutants, and for general reversions (Kondo et al., 1970), appear to be significantly increased. Loop formation opposite persisting gaps in W3110 polAl has been suggested to account for the increased spontaneous frequency of deletion mutants (Okazaki et al., 1971). However, other groups have found essentially no difference in the rate of mutagenesis between W3110 polAl and its parent or a polA + revertant (Witkin, 1971 ; Smirnov et al., 1971 ; Berg, 1971). Several types of evidence indicate that W3110 polAl cells are defective in an excision repair system. A greatly increased sensitivity to X-irradiation (Town et al., 1971 ; Paterson et al., 1971), to MMS (Gross and Gross, 1969; Kato and Kondo, 1970; Smirnov et al., 1971), and to caffeine (Bahr and Schuster, personal communication), can be measured either

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by clone-forming ability, extent of DNA degradation or curtailment of DNA synthesis. The mutant also exhibits a somewhat increased sensitivity to UV-irradiation (Gross and Gross, 1969; Kato and Kondo, 1970; Boyle et al., 1970; Kanner and Hanawalt, 1970; Paterson et ak, 1971; Monk et al., 1971), and is unusually sensitive to thymineless death (J. Cairns, personal communication; Smirnov et al., 1971). The degree of increased sensitivity to UV-irradiation appears to be greater for stationary phase cells than for exponentially-growing cells (Green et al., 1971a)in a polymerase-deficient E. coli B resA mutant (Kato and Kondo, 1970), in contrast to E. coli TAU-bar (Hanawalt, 1965). W3110 polAl also exhibits a reduced ability to support the growth of UV-irradiated phage (Kato and Kondo, 1970; S. Smith et al., 1970; Paterson et al., 1971; Klein and Niebch, 1971). The deficiency appears to be in a "rapid-repair" system for both X-ray damage (Town et aL, 1971) and UV-induced damage (Cooper and Hanawalt, 1972a, b). It is possible that DNA polymerase I functions in this repair normally as either (1) a polymerase by filling gaps in one of the two DNA strands of a native double-stranded DNA molecule (Fig. 14B), or (2) as a nuclease or combined nuclease-polymerase by generating and/or filling in such gaps (for example, as observed by Kelly et al., 1969), or (3) by nick-translation without generation of a gap (Fig. 14E). W3110 polAl can excise thymine dimers (Boyle et al., 1970), and rejoins DNA strands following UV-irradiation (Kanner and Hanawalt, 1970) or MMS-treatment (Sinzinis et al., 1971) more slowly than does the parent polA ÷, consistent with the above possibilities. Properties of the isolated enzyme, and its possible role in DNA replication, are discussed below (sections IIIC and IIIE). In addition to the excision-repair system, wild type E. coli possesses a recombination system for repair of damaged DNA (Howard-Flanders et al., 1968; Smith and Meun, 1970). Attempts to isolate a recApolA double mutant have been unsuccessful (Gross et al., 1971); such mutants may be nonviable. This would be expected if DNA polymerase I is needed for the excision-repair system, and the recA gene product is required for the recombination repair system, and if the cell must possess one of these two repair systems to survive. However, u v r - r e c A double mutants are viable, and the uvr loci are thought to have gene products involved in excision repair. It may be that the cell must have either the "'rapid repair" system or the recombination repair system to survive, and that the uvr gene products are dispensable for the "rapid repair" system. Repair of gaps (Monk et aL, 1971) and "delayed" repair of X-ray induced damage (Kapp and Smith, 1970) in W3110 polAl appear to be due to the recombination repair system. Kuempel and Veomett (1970) and Okazaki et al. (1971) have observed that pulse-labeled DNA in W3110 polAl cells persists as short fragments when compared with pulse-labeled DNA in polA ÷ cells, suggesting a possible role of DNA polymerase I in DNA replication when DNA polymerase I is present in the cell. This role may be that of a repair-type synthesis, necessary and normal during DNA replication. (b) D N A polymeraxe 111. Recently, two additional DNA polymerase activities, called DNA polymerase I[ and DNA polymerase III, have been isolated from W3110 polAl cells (Knippers, 1970; Kornberg and Gefter, 1970; Moses and Richardson, 1970a, b; Kornberg and Gefter, 1971). In an attempt to correlate enzymatic activities with conditional mutants deficient in DNA synthesis, the temperature-sensitive DNA mutants (section IIIB) have been screened at the restrictive temperature for enzymatic activities possibly relevant to DNA replication (for example, see Bonhoeffer and Messer, 1969). The first positive correlation has only recently been reported. Gefter et al. (1971) have found that the activity of the DNA polymerase II[ isolated from 2 of 4 dnaE mutants examined at 45 ° compared with that

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at 30 ° was dramatically reduced. No similar thermosensitivity was observed with the isolated DNA polymerase III from other dna mutants, nor with the isolated DNA polymerase II from any dna mutants. It appears that the structural gene for DNA polymerase III is the mutated gene from these 2 dnaE mutants. DNA polymerase III thus appears to be the first E. coli enzyme that can be said to be directly involved in DNA replication. The fact that not all dnaE mutants contain a thermosensitive polymerase III could be due to several reasons. For example, more than one gene leading to thermosensitive dna mutants could be located in the dnaE region (see Fig. 7). Alternatively, some mutants in the polymerase III gene may lead to polymerase III molecules which are thermosensitive in vivo, but exhibit a relatively temperature-independent activity in the in vitro assay reactions. This latter possibility is suggested by the complementation experiments of Niisslein et al. (1971). Using the cellophane-membrane in vitro DNA synthesis system (Schaller et al., 1972) with extracts of dnaE mutants, Niisslein et al. (1971) have isolated an enzymatic activity which complements the in vitro synthesis system at the restrictive temperature. This isolated activity is indistinguishable in its properties in a typical in vitro assay from the DNA polymerase III of Kornberg and Gefter (1971). The activity has different properties when assayed in the usual in vitro reaction using activated DNA as a substrate (see, for example, Richardson, 1967) and when assayed in the cellophane-in vitro DNA synthesis system. In the former, activity is low and inhibited by KCI; in the latter, activity is about ten-fold higher, and is salt independent (Niisslein et al., 1971). This observation also exemplifies a general concern that enzymatic properties need to be determined in an environment and with substrate molecules very similar to those found within the cell. The in vitro DNA synthesis systems (see section IIID) hopefully will provide this possibility. (c) D N A ligase. Pauling and Hamm (1968), using the BU-incorporation selection method of Bonhoeffer and Schaller (1965), modified to select for temperature-sensitive, UVsensitive and repair replication deficient mutants, isolated a mutant of E. coli TAU-bar called ts-7 which exhibits a two-fold increased sensitivity to UV-irradiation at 40 ° compared to that at 27 °. Examination of pulse-labeled DNA showed that the short fragments synthesized at 40 ° are not joined together, suggesting a thermo-sensitive DNA ligase. Although initial experiments (Pauling and Harem, 1969a; Gellert and Bullock, 1970) indicated reduced ligase activities at both temperatures, with little temperature dependence, subsequent studies (Modrich and Lehman, 1971), using different ligase assays, have demonstrated the presence of a thermosensitive DNA ligase in ts-7. Since ts-7 cells die when incubated at the high temperature, it would appear that ligase activity is indispensable to the cell. However, the cells do not die immediately, and DNA synthesis continues, although at a reduced rate. DNA synthesized in the presence of BU has a lower buoyant density than is normally found (Pauling and Hamm, 1968), indicating an abnormal DNA synthesis, perhaps a nick translation synthesis by D N A polymerase I at nicks left by the absence of DNA ligase (Fig. 14E). Because ts-7 is a derivative of E. coli TAU-bar, it has not been genetically mapped. Using a two-step isolation procedure at 37 °, Gellert and Bullock (1970) have isolated several mutants of E. coli K12 containing reduced amounts of DNA ligase. Ligase "overproducing" strains, called lop mutants, capable of supporting growth of gene 30 phage T4 mutants lacking a functional T4 DNA ligase gene (see Table 3) were isolated first. One of these, lol)8, produced five times as much ligase as wild-type cells, each molecule of which was like wild-type ligase. This strain was used to reisolate mutants incapable of supporting the growth of the ligase-deficient T4 phage; three of these mutants produced defective ligase P.e. 26.--M

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molecules. One of these, lop8 lig4, was strongly thermosensitive, exhibiting wild-type activity at 30 ° and 470 activity at 42 °. Although they are somewhat UV-sensitive, growth of these mutants was essentially normal, and the short DNA pieces do not accumulate, in contrast to ts-7. However, each of these strains exhibit some (2-10~) residual ligase activity, and this may be sufficient to perform a putative indispensable ligase function in D N A replication. There is also the possibility that the ligase activity of these cells is normal in vivo although reduced in vitro. Formation of the AMP-enzyme complex appears to be normal. Mutants with less residual activity have recently been isolated (Gellert and Hicks, personal communication). The best, having < 170 of normal in vitro ligase activity at 42 °, grows normally at 42 °, but the rate of joining of the Okazaki fragments is reduced five- to tenfold. Both lop and lig loci have been mapped near the ctr locus on the E. coli map (Fig. 7). (d) Nuclease mutants. Mutants of E. coli deficient in endonuclease I (and ribonucleases I and II) have been isolated using Giemsa (Diirwald and Hoffmann-Berling, 1968) or methyl green (Wright, 1971) staining of toluene-treated colonies. Mutant colonies unable to selfdegrade their DNA (or RNA) stain differently from wild-type colonies. E. coli cells deficient in endonuclease I activity grow normally under all growth conditions, and support the growth of all bacteriophage tested. Mutant cells are further indistinguishable from wildtype endoI ÷ cells with respect to recombination sensitivity of spheroplasts to fd-DNA infection, and host-controlled restriction towards phage fd (Diirwald and HoffmannBerling, 1968). Diirwald and Hoffmann-Berling (1968) mapped the locus for these mutations near str at about 65 min (see Fig. 7). In addition to this endoA locus, Wright (1971) has found another, endoB, mapping between 13 and 25 min on the E. coli genetic map. Appearance of newly replicated DNA as Okazaki fragments in endoI- cells is similar to that of endoI ÷ cells (Okazaki et al., 1968b). The intracellular role of DNA endonuclease I remains obscure. EndoI ÷ cells, when treated with EDTA, incorporate dNTP's in a nonconservative mode of DNA synthesis (Buttin and Kornberg, 1966). However, endoI- cells do not exhibit this synthesizing ability (Buttin and Wright, 1968), suggesting that endonuclease I, in the EDTA-treated wild-type cells, introduces nicks into the DNA which can serve as primers for synthesis by DNA polymerase I. Further, an ATP-independent nonconservative repair-type synthesis is observed in toluene-treated poIA÷endoI ÷ cells (see section IIID) which is not observed in p o l A ÷ e n d o I - cells (Moses and Richardson, 1970a). Recombination-deficient mutants ofE. coli have been isolated which map at three different loci (see Fig. 7): recA, recB, and recC (Clark and Margulies, 1965; Howard-Flanders and Theriot, 1966; Clark, 1967). R e c B and recC mutants fall into two separate complementation groups, although they both cotransduce with t h y A and a r g A (Emmerson and HowardFlanders, 1967; Emmerson, 1968; Willets and Mount, 1969). These mutants are very sensitive to X-irradiation, but are less sensitive to UV-irradiation than the recA mutants (Howard-Flanders and Boyce, 1966). They exhibit little spontaneous DNA degradation during growth, are able to induce phophage lambda normally (Clark, 1967), and fail to form recombinants. An ATP-dependent deoxyribonuclease, detectable in rec ÷ cells, is absent from recB or recC cell extracts (Buttin and Wright, 1968; Oishi, 1969; Barbour and Clark, 1970; Goldmark and Linn, 1970; Wright et al., 1971). The enzymatic properties of this enzyme, exhibiting both properties of an exonuclease and an endonuclease, as well as a DNA-dependent ATPase (Nobrega et al., 1972), are discussed below (section IIIC), and the role of the recB gene product in the in vitro DNA synthesis systems is discussed in section

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IIID. Recently, double mutants of recB and recC strains have been obtained in which recombination proficiency is restored. The second mutations are found in two loci called sbcA and sbcB (indirect suppression of recB, recC). SbcBrecB double mutants have decreased activity of an exonuclease specific for denatured DNA; purification and immunological data has shown this nuclease to be exonuclease I (Kushner et al., 1971). SbcA mutants may have increased activities of a new ATP-independent deoxyribonuclease (Barbour et al., 1970). 2. Bacterial mutants: temperature-sensitive dna mutants Selection methods which preferentially kill wild-type cells able to synthesize DNA at a restrictive temperature, either high or low, have been developed for isolation of temperaturesensitive DNA synthesis mutants. Such methods include killing of cells which have incorporated BU during DNA synthesis at the restrictive temperature with near UV-irradiation (Bonhoeffer and Schaller, 1965) and death due to radioactive decay of incorporated aHthymidine (Fangman and Novick, 1968). In addition, many temperature-sensitive mutants have been screened for specific temperature-sensitive DNA synthesis mutants (Kohiyama et al., 1966; Kohiyama, 1968; Gross et al., 1968; Kuempel, 1969; Carl, 1970; Karamata and Gross, 1970). Properties of these mutants have been recently reviewed (Gross, 1972); the following will present some of the more general points. Thermosensitive mutants for DNA replication have also been isolated from B. subtilis (Gross et al., 1968; Bazill and Retief, 1969; Boylan and Mendelson, 1969; Yoshikawa, 1970a; Mach and Engelbrecht, 1970; Karamata and Gross, 1970; Gross, 1972; Matsushita et al., 1971) and in S. typhimurium (Spratt and Rowbury, 1970a, b). The dna mutants orE. coli fall into seven major groups according to their genetic and biochemical properties; mutants in each group are termed dnaA, dnaB, dnaC, dnaD, dnaE, dnaF, and dnaG mutants. Their approximate map positions (Fig. 7) have been determined using Hfr and F-prime conjugation, and phage PI transduction methods (Hirota et al., 1968a; Fangman and Novick, 1968; Carl, 1970; Hirota et al., 1970; Mordoh et al., 1970; Gross, 1971 ; Hirota et al., 1972; Wechsler and Gross, 1972). The mutants are basically of two types: those which cease DNA synthesis immediately when placed at the restrictive temperature, and those which exhibit some residual synthesis before cessation. The former, "immediate shut-off" mutants, include dnaB, dnaD, dnaE, and dnaG mutants; the latter, possibly involved in initiation, include dnaA and dnaC mutants. DnaF mutants show some residual DNA synthesis, but less than dnaA or dnaC mutants (Wechsler and Gross, 1972). Several enzyme activities have been assayed in some of these strains. None tested, including many dnaB mutants from Bonhoeffer's collection, some dnaA, and a few dnaG mutants, are deficient in DNA polymerase [ (Bonhoeffer and Messer, 1969; Gross, 1972; Wechsler and Gross, 1972). No dnaB mutants possess temperature-sensitive ligase activities. Recently, the isolated DNA polymerase III from some dnaE cells has been shown to be temperature-sensitive (Gefter et al., 1971), and an activity indistinguishable from polymerase III is able to supply the deficient function using dnaE cells at 42° in the in vitro cellophane DNA synthesis system (Niisslein et al., 1971). No other enzymatic activities associated with the temperature-sensitive genes have been identified. The T-even phages are propagated by all E. coli dna mutants at 42°, whereas phage lambda can use only the dnaA and dnaC residual synthesis mutants as hosts at 42 ° (Fangman and Novick, 1968; Hirota et al., 1968a, b; Inouye, 1969; Fangman and Feiss, 1969; Lanka and Schuster, 1970; Carl, 1970; Beyersmann et al., 1971). When dnaB70 mutants are

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shifted to 42 ° early after infection at 33 ° with phage lambda, lambda D N A synthesis, as well as that of the host, ceases immediately. However, when shifted to 42 ° late after infection, some residual lambda D N A synthesis occurs, with some indication that replicating lambda D N A molecules complete a round of replication (Mikolajcyk and Schuster, 1971). Spontaneous induction of lambda lysogens of dnaAS08 when placed at 43 ° does not occur. However, such lysogens are UV-inducible, even after completion of the residual DNA synthesis at 43 ° and in cells lacking the ucr gene products. Thus, induction can occur in cells following UV-damage in the apparent absence of DNA replication (Monk and Gross, 1971). Phage P1 can use dnaB70 as host, but not dnaA46, at 42 ° (Lanka and Schuster, 1970; Beyersmann et aL, 1971). P1 mutants have been isolated which, as prophage, suppress the dnaB defects (Jaffe-Brachet et ak, pers. comm.). The single-stranded DNA phages ~ × 174 and M13 are not propagated by dnaB mutants at 42°; in fact, the single-stranded phage D N A is not even converted into the parental RF, although it does enter the cell (Steinberg and Denhardt, 1968; Sinsheimer et al., 1968; Primrose et al., 1968). If this initial step in phage D N A synthesis is permitted at low temperature before incubation at 42 °, again no phage are produced, indicating that a later step in D N A synthesis is also blocked in these mutants. However, after beginning synthesis at the low temperature, M13 can continue single-strand D N A synthesis for about 2 hr at 42 °, whereas ~ × 174 single-strand synthesis continues for only a few minutes in similar experiments (Steinberg and Denhardt, 1968). In a dnaA mutant (E. coli ts 120/6), the plasmid ColEl D N A synthesis ceases immediately at 43 °, whereas in the dnaB43 mutant, synthesis continues at a reduced rate, generating abnormal large forms (Goebel, 1970a). If the dnaA mutant is indeed an initiator mutant for the E. co/i replicon, the temperature-sensitivity of ColEl, as a second independent replicon, is difficult to explain. In the dnaB43 mutant, both E. co/i and ColEl D N A are degraded slowly at 43 ° and the newly synthesized E. coli D N A is preferentially degraded. However, pulse-labeled ColE1 DNA, in contrast, is reportedly specifically resistant to degradation at 43 ° (Goebel, 1970b). The dnaB43 mutant also has the ability at 31 ° to permit the entry of an F-prime factor into a cell containing another F-prime factor, and to permit stable intracellular coexistence of the two. Membrane alterations are proposed to explain this observation (Palchoudhury and Iyer, 1971). It is clear from these initial observations that the dna mutants promise to provide much information about phage D N A replication, and conversely that the study of phage D N A replication in these mutants may provide information about the mutants themselves. DnaA and dnaB mutants lose viability at the restrictive temperature in an unknown manner dependent on metabolic activity (Fangman and Novick, 1968; Buttin and Wright, 1968; Couch and Hanawalt, 1967; Lanka and Schuster, 1970; Carl, 1970). Upon return to the permissive temperature after a limited time at the restrictive temperature, nearly all dnaB mutants recover quickly (Inouye, 1969; Ricard and Hirota, 1969) in the absence or presence of protein synthesis (Kogoma and Lark, 1970; Worcel, 1970). However, upon return to the permissive temperature, premature initiation of a new round of replication, as well as continuation at the old growing point, occurs (Stein and Hanawalt, 1969; Inouye, 1969; Ricard and Hirota, 1969; Kogoma and Lark, 1970; Schwartz and Worcel, 1971). As is observed following thymine starvation (Pritchard and Lark, 1964; Kallenbach and Ma, 1968) or nalidixic acid treatment (Boyle et al., 1967; Ward and Glaser, 1970), only one of the two daughter D N A chromosomes initiates a premature round of replication (Stein and Hanawalt, 1969; Shapiro et al., 1970; Worcel, 1970). This premature initiation occurs in all cells following only 10 rain incubation at the high temperature, apparently at a unique

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chromosomal site identical to that observed in amino acid starvation experiments (Schwartz and Worcel, 1971). No premature initiation is observed in recA/dnaBT266 double mutants (Worcel and Schwartz, pers. comm.). Perhaps the recA gene product is needed for a recombination event involving daughter DNA and parental DNA found at the terminus (Yoshikawa, 1967, 1970a; Stein and Hanawalt, submitted for publication). Further, only one new round of replication occurs after incubation at the high temperature, rather than the premature initiation of several rounds of replication observed following thymine starvation or nalidixic acid treatment (Kogoma and Lark, 1970). DnaA mutants (dnaA46 and dnaA83 and dnaC mutants (dnaC2) recover at the low temperature after limited incubation at the high temperature (Worcel, 1970; Beyersmann et al., 1971). DnaB22 mutant cells appear to be X-ray sensitive, but not UV-sensitive, in a temperaturedependent manner (Fangman and Russel, 1971). Following UV-irradiation at 42 °, dimer excision, DNA breakdown, repair replication, and joining are all normal. However, following X-irradiation at the restrictive temperature, no repair is observed, and considerable DNA degradation occurs. This suggests the presence of separate pathways, at least in part, for repair of UV-induced and X-ray damage. The effects of the dna mutations in some dna mutants can be phenotypically reversed with high salt, glucose, or glycerol at the high temperature. That is, the phenotypically reversed mutants grow at the restrictive temperature in the presence of the small molecule. Several dna mutants apparently synthesize DNA normally at 42° in 0.2~o to 2~o NaC1, and cell division occurs in the presence of 0.2~o to 2~o NaCI plus glucose (Ricard and Hirota, 1969; Hirota et al., 1972). Gross (1972) observed that all dnaB and dnaE mutants examined were phenotypically reversed by both high glucose and high glycerol concentrations. DnaA mutants were phenotypically reversed by high glucose concentrations but not by high salt. In another study of 54 independent thermosensitive DNA synthesis mutants, 30 were phenotypically reversed by either 2~o NaC1 or 20~ sucrose, 8 with NaCI but not by sucrose, 7 by sucrose but not by NaC1, and the remaining 19 were not phenotypically reversible (Hirota et al., 1972). It is clear that these effects are complex and probably the result of several physiological events. None of the E. coli mutants examined appear to be deficient in precursor synthesis, although B. subtilis mutants defective in the reduction of ribonucleotides exist (Karlstrom, 1971). Repair replication, examined in the dnaB43 mutant, appears to be normal (Couch and Hanawalt, 1967). Recombination ability appears normal, as assayed by testing for alkaline phosphatase activity in Hfr-F- zygotes following transfer of a wild-type alkaline phosphatase structural gene (pho +) into apho-dnaB43 recipient (Joshi and Siddiqi, 1968). E. coli dna mutants have been used in studies of DNA synthesis in the recipient and/or the donor during bacterial conjugation. The conflicting results from these and other studies in this controversial area of research have been recently reviewed and authoritatively discussed (Curtiss, 1969). It now appears from the elegant physical studies of Vapnek and Rupp (1970) that DNA synthesis occurs in both the donor and in the recipient during Hfr or F-prime transfer; these studies were discussed above. One possible problem with early studies was the use of nonisogenic strains. Marinus and Adelberg (1971) have recently shown that transfer occurs normally between recipient and donor cells when each contain the same dna mutation, using both dnaB and dnaG mutants. F factor mediated DNA replication thus apparently does not require the missing functions in these strains. Analysis of isolated F-factor supercoiled DNA from recipient and donor cells has led to similar results (Vapnek and Rupp, 1971). Further, Stallions and Curtiss (197l) observed normal

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DNA transfer using a variety of dnaB recipient and donor strains, but found a requirement for the dnaB gene product in the recipient cell to yield recombinants, consistent with the earlier results of Bonhoeffer (Bonhoeffer, 1966; Bonhoeffer et aL, 1967). Much circumstantial evidence suggests that at least some dnaA and dnaC mutants are initiation mutants (Hirota et aL, 1970; Carl, 1970; Beyersmann et al., 1971 ; Hirota et al., 1972; Wolf, pers. comm.). The amount of residual synthesis at the high temperature is comparable to that observed at the permissive temperature in the absence of protein synthesis, suggesting completion of rounds of replication at the restrictive temperature (Beyersmann et aL, 1971 ; Hirota et aL, 1970; Carl, 1970). No residual synthesis at 42 ° is observed if completion of rounds of replication is first permitted via protein synthesis inhibition at the permissive temperature (Beyersmann et al., 1971 ; Hirota et aL, 1970). As mentioned above, upon return to the permissive temperature, reinitiation occurs, resulting in synchronized DNA replication at a site indistinguishable from the site of initiation following protein synthesis inhibition (Hirota et al., 1970; Carl, 1970; Beyersmann et al., 1971). The temperature-sensitive gene products of the dnaA and dnaC mutants remain unknown. However, Wechsler et aL (1972) and Carl (1970), using appropriate F-prime merodiploids, have shown that the mutations in many dnaA mutants and in dnaC1 and dnaC2 are recessive, as expected for a diffusible gene product. Upon return to the permissive temperature, three mutants (dnaC28, dnaA252, and dnaC325) rapidly initiate a round of replication even in the presence of 25 /~g/ml chloramphenicol, suggesting reversible denaturation of the gene products involved. However, three other mutants (dnaA83, dnaA46, and dnaA47) show no such initiation in the absence of protein synthesis upon return to the low temperature, indicating irreversible denaturation of the gene products (Gross, 1972). The variation in properties among dnaA and dnaC mutants suggest that some of the mutant groups comprise more than one gene. The existence of "integrative suppression" further implicates the dnaA and dnaC thermosensitive gene products in initiation. F + derivatives of dnaA mutants and of dnaCl give rise to temperature-resistant"revertants" at a high rate. These new strains have integrated the F-factor into the bacterial chromosome (Nishimura et aL, 1971). Presumably the F-factor, as an independent replicon (Jacob et aL, 1963), contains genetic information for its own initiation system. Replication of the bacterial chromosome would hypothetically proceed passively under regulation of the F-factor replicon (Thomas and Mousset, 1970). However, it has not been shown that the replication observed at the high temperature proceeds from the F-factor site as origin. As another example of "integrative suppression", phage P2 mutants exist (P2sig) which permit host DNA replication in dnaA46 at 42 ° when present as the prophage (Lindahl et aL, 1971; Hirota et al., 1972). Thermoresistance effected by the suppression via integration of P2sig occurs only when P2sig is integrated near metE; the host, however, has lemained dnaA46 (see Fig. 7). The gene A product of phage P2 is required for integrative suppression, but the gene B product is not. Upon shift to the restrictive temperature, DNA degradation is observed in dnaB, dnaD, and dnaG mutants (Buttin and Wright, 1968; Carl, 1970; Gross, 1972; Mikolajczyk and Schuster, 1971), but not in dnaA or dnaC mutants (Hirota et al., 1968b; Carl, 1970). This degradation is enhanced by the ATP-dependent DNase (Buttin and Wright, 1968), the reeB gene product (Oishi, 1969; Goldmark and Linn, 1970; Barbour and Clark, 1970; Oishi, 1970). In dnaB mutants, newly replicated DNA is preferentially degraded (Rasmussen, 1968; Mikolajczyk and Schuster, 1971). The electrophoretic properties of one or a few cell membrane proteins are altered in both

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351

dnaA and dnaB mutants at the restrictive temperature (Inouye and Guthrie, 1969; Inouye and Pardee, 1970; Shapiro et al., 1970; Siccardi et al., 1971) and in dna + cells grown under thymine-starvation conditions (Inouye and Pardee, 1970; Siccardi et aL, 1971). Whether

these changes cause or result from cessation of DNA synthesis is unknown. However, dnaB mutants phenotypically reversed by high salt exhibit membrane protein alterations, even though their DNA synthesis is supposedly normal (Siccardi et al., 1971). Less direct evidence also suggests membrane or cell wall changes. Hirota et aL (1972) found that dnaA46 and dnaA83 cells, after incubation at the high temperature, were more sensitive to lysis

by deoxycholate and more readily bound an aniline dye. Kingsbury et al. (1972) have observed similar sensitivity to deoxycholate in their temperature-sensitive polA mutants ts214 and ts216, as well as in polA1 itself. As is true with any treatment which specifically inhibits DNA synthesis, e.g., thymine starvation or UV-irradiation, most dna mutants at the restrictive temperature do not divide, but elongate, forming very long "snakes" (Bonhoeffer and Schaller, 1965; Hirota et aL, 1968a, b; Inouye, 1969). "Snakes" are also often observed in broth cultures of W3110 polA1. However, some dnaA mutants do divide at the higll temperature, forming anucleate cells (Hirota et al., 1968b; Inouye, 1969). These mutants may carry a second mutation resulting in this uncoupling of cell division from DNA replication (Hirota et al., 1968a). 3. Bacteriophage mutants

Because of space considerations, only a few selected examples are considered here. The following are a few of the many recent books and reviews on this general topic: Stent, 1963; Luria and Darnell, 1968; Fraenkel-Conrat, 1968; Colter and Paranchych, 1967; Fenner, 1968; Cohen, 1968; Cold Spring Harbor Symposia, 1968, 1969, 1970; Calendar, 1970. (a) T4. The genome of the T-even phages is a single DNA molecule, circularly permuted and terminally redundant(Thomas and MacHattie, 1967) having a molecular weight of about 120 × 106 daltons (Burgi and Hershey, 1963). This large DNA molecule, about one-twentieth the size of the E. coli chromosome (Cairns, 1963a) and one-fourth that of Mycoplasma hominis (Bode and Morowitz, 1967), contains information for about 200 genes. Approximately 90 genes, many essential as defined by conditional lethal mutations (Epstein et al., 1963), have been identified and are shown in Fig. 12. Synthetic abilities following infection of E. coli with these mutants and the properties of the defective phage particles and components have been extensively studied and classified (Epstein et al., 1963; Kozloff, 1968; Cohen, 1968; Radding, 1969; Koerner, 1970; Calendar, 1970). Mutants defective in DNA synthesis are shown in Table 1. Some of the enzymatic activities have been identified, the enzymes purified, and their properties characterized. Properties and possible functions of the T4 DNA polymerase (gene 43 product), the T4 ligase (gene 30 product), the T4 denaturation protein (gene 32 product), and phage-induced DNases are discussed in sections IIIC and IIIE. The nucleotide composition of the DNA is unusual in that cytosine residues are replaced by 5-hydroxymethylcytosine residues (Wyatt and Cohen, 1953), which are further modified by the T-even glucosylation system (Luria, 1953; Hattman and Fukasawa, 1963). The enzyme systems involved have been extensively studied and reviewed (Koerner, 1970; Cohen, 1968). Shortly after infection, host DNA, RNA, and protein synthesis cease (Kaempfer and

352

DOUGLAS W. SMITH

Magasanik, 1967; Landy and Spiegelman, 1968; Hosoda and Levinthal, 1968; Adesnik and Levinthal, 1970). The mechanisms remain unknown, although phage protein synthesis is required for most of these steps (Hayward and Green, 1965; Ennis, 1970; Adesnik and Lcvinthal, 1970). Host D N A is degraded to monodeoxynucleotides (Wiberg et al., 1962; Wiberg, 1966); this degradation is blocked by mutations in phage genes 46 and 47, suggesting that one or both of these genes code for a nuclease, perhaps specific for cytosinecontaining D N A (Wiberg, 1966). Two T4-induced nucleases, endonuclease II and endonuclease IV (see section IIIC), one of which recognizes cytosine residues and neither of which degrade T4 DNA, have been purified (Sadowski and Hurwitz, 1969a, b). Mutants for endonuclease II have been isolated (Warner et al., 1970; Hercules et al., 1971) and map between genes 32 and 63.

55 49

rt

J

30

c

52

-tdcd 63

31

FIG. 12. Genetic map of bacteriophageT4. Narrow genes: early regions of the chromosome for gene expression. Wide genes: late regions of the chromosome for gene expression. Dark genes: those involved in phage morphogenesis. The arrows denote direction of translation, as determined by polarity of mutations (open arrows) or by amino acid or protein sequencing (closed arrows). Compiled by M. V. Sheldon, Ph.D. Dissertation, University of Chicago, 1972.

The gene 42 product, dCMP hydroxymethylase, in addition to catalysis of dCMP hydroxymethylation, may play a direct role in phage D N A synthesis (Chiu and Greenberg, 1968; Scotti, 1969). Gene 42 mutants excrete acid-soluble host DNA breakdown products (Wiberg et al., 1962). A source of dCMP is provided by the gene 56 product, deoxycytidine triphosphatase, which catalyzes the hydrolysis of dCTP to dCMP and PP~ (Kornberg et al., 1959; Koerner et al., 1959, 1960). Gene 56 mutants initiate D N A synthesis at a normal rate, but synthesis rapidly ceases and the synthesized D N A is degraded (Wiberg, 1966; Warner and Hobbs, 1967; Kutter and Wiberg, 1968), suggesting that a phage-induced nuclease

D N A SYNTHESIS IN PROKARYOTES : REPLICATION

353

degrades the cytosine-containing DNA. However, the DNA can contain as much as 20~o cytosine (Wiberg, 1967), and yield viable phage. Glucosylation of the DNA is not necessary to render the DNA resistant to nuclease attack (Wiberg, 1967). Although gene 46 and 47 mutants are deficient in host DNA breakdown, recent evidence suggests that these genes control a nuclease which can attack T4 DNA (Cascino et aL, 1970; Hosoda and Mathews, 1971 ; Kutter and Wiberg, 1968), and that this nuclease may be involved in genetic recombination (Baldy, 1968; Bernstein, 1968). Shah and Berger (1971) and Shalitin and Naot (1971) have examined the fate of parental T4 DNA from gene 46,47 double mutants. The parental DNA sediments rapidly, as does wild-type T4 parental DNA (Frankel, 1966b; Frankel et al., 1968), but the early DNA replication complex is unstable and dissociates prematurely. Further, DNA isolated from cells late in infection acquires discontinuities, a property which is prevented by 100/zg/ml chloramphenicol (Shah and Berger, 1971). This short DNA is converted into nonviable phage particles deficient in DNA content (Shalitin and Kahana, 1970). The evidence suggests that only attached DNA can replicate (Shalitin and Naot, 1971); the "DNA arrest" phenotype of gene 46,47 mutants could thus be attributed to premature release from the replication site. Shalitin and Naot (1971) proposed that gene 46 controls an exonuclease which participates in attachment of nicked parental DNA to the membrane site via a recombination event. The phenotype of the gene 46 mutants can be suppressed by a second mutation in gene 33 (Shalitin and Naot, 1971); this second mutation is possibly the same as the das mutations (DNA arrest suppressor) which specifically suppress gene 46 and 47 mutations and map in gene 33, or between genes 33 and 34 (Hercules and Wiberg, 1971). Mutants in gene 30 lack the T4-induced DNA ligase (Fareed and Richardson, 1967), and thermosensitive gene 30 T4 mutants appear to accumulate the Okazaki fragments during infection at the restrictive temperature (Okazaki et aL, 1968b; Newman and Hanawalt, 1968a, b; Hosoda and Mathews, 1968). However, this accumulation may be due to a T4induced nuclease activity, as suggested by Kozinski (1968), rather than to a reduction in ligase activity (Hosoda and Mathews, 1971). The identity of the putative nuclease remains unknown. However, when gene 30 T4 mutants are rendered double mutants by introduction of an rII mutation, the effects of the gene 30 mutation are suppressed, restoring DNA synthesis and phage propagation (Berger and Kozinski, 1969; Karam, 1969; Chan et al., 1970). A similar suppression of the gene 30 mutation occurs when gene 30 mutants are initially grown in the presence of chloramphenicol (Berger and Kozinski, 1969; Hosoda and Mathews, 1971 ; Iwatsuki and Okazaki, 1970). These results are interpreted to mean that a nuclease activity, reduced in rII mutants or by chloramphenicol treatment, must be limited during normal phage growth by T4 ligase. The rII gene product may thus be a nuclease. Gene 30 mutants also resume some host DNA synthesis, yielding an unstable DNA product. This reaction appears to require E. coli DNA polymerase I, but not the products of T4genes 32, 41, 43, 44, or 45, and appears to be an attempt at DNA repair. The T4 DNA ligase may thus also function in host DNA degradation (Cascino et al., 1971a). The product of gene 32, the renaturation-denaturation protein (Alberts et aL, 1968; Alberts and Frey, 1970), is required throughout the T4 lytic cycle. DNA synthesis stops immediately when temperature-sensitive gene 32 T4 mutants are shifted to the restrictive temperature during infection (Alberts et aL, 1968). Further, the quantity of gene 32 protein synthesized during infection directly limits the burst size of progeny phage (Snustad, 1968; Sinha and Snustad, 1971), even though about 10,000 molecules of this protein are synthesized per cell in the T4 lytic cycle (Alberts, 1970). Thus, the gene 32 protein is

354

DOUGLAS W. SMITH TABLE I PROPERTIES OF T4 MUTANTS DEFECTIVEIN D N A SYNTHESIS

Gene number

Phenotype classification

Enzyme deficiency

1

DO

Deoxynucleoside kinase

30 32 33

DA DO MD

DNA ligase Denaturation protein Unknown

39 41 42

DD DO DO

Unknown Unknown dCMP hydroxymethylase

43

DO

DNA polymerase

44 45 46 47 52 55 56

DO DO DA DA DD MD DO

Unknown Unknown Nuclease (?) Nuclease (?) Unknown Unknown dCTPase-dUTPase

59 60 61 62

DA DD DD DO

Unknown Unknown Unknown Unknown

DO: DA: DD: MD:

References Epstein et aL, 1963; Duckworth and Bessman, 1967 Fareed and Richardson, 1967 Alberts et al., 1968 Epstein et aL, 1963; Pulitzer and Geiduschek, 1970 Yegian et al., 1971 Epstein et al., 1963; Oishi, 1968c Wiberg et al., 1962; Dirksen et al., 1963; Wiberg and Buchanan, 1964 DeWaard et al., 1965; Warner and Barnes, 1966 Epstein et al., 1963 Epstein et al., 1963 Hosoda and Mathews, 1971 Hosoda and Mathews, 1971 Yegian et aL, 1971 Pulitzer and Geiduschek, 1970 Wiberg, 1967; Munro and Wiberg, 1968; Price and Warner, 1968 Wu et al., 1972 Yegian et al., 1971 Yegian et aL, 1971 Karam, personal communication

no DNA synthesis. DNA synthesis arrested. DNA synthesis delayed. maturation defective.

required in stoichiometric amounts and hence acts like a structural protein. Further, its function is necessary for the formation of the hydrogen-bonded D N A molecules found during recombination (Tomizawa, 1967). Since D N A replication, is not obligatory for recombination to occur in T4-infected E. coli (Tomizawa et al., 1966), the gene 32 protein appears to be necessary for both T4 D N A replication and T4 D N A recombination (Alberts and Frey, 1970). Phage T4 D N A found early in infection differs from mature T4 D N A in several respects. It is unusually large (Frankel, 1963; 1966a, b; Frankel et al., 1968; Kozinski et al., 1967; Streisenger et al., 1967; Altman and Lerman, 1970a, b), and in part is found in a protein complex (Frankel et al., 1968; Miller and Kozinski, 1970a; Miller and Buckley, 1970; Altman and Lerman, 1970a). Continued T4 D N A replication is necessary for late transcription (Mathews, 1968; Lembach et al., 1969; Riva and Geiduschek, 1969; Riva et al., 1970a, b; Cascino et al., 1970, 1971a; Shalitin and Kahana, 1970); mature T4 D N A is incompetent for this task (Cascino et al., 1970). This "coupling" between continued D A N replication and late transcription is retained when the D N A is not glucosylated (Sauerbier and Brautigam, 1970; Riva et al., 1970b), but is not retained in gene 30-gene 46 double mutants (Riva et al., 1970b). Since the gene 46 mutation simply prevents degradation of the unligated DNA, these results suggest that D N A competent for late transcription contains nicks or breaks, and that ligase controls late transcription in a negative manner.

D N A SYNTHESISIN PROKARYOTES: REPLICATION

355

The gene 33 and 55 products are also required for late transcription (Adesnik and Levinthai, 1970; Cascino et al., 1971a). These gene products are necessary for the shut-off of preearly transcription (Sk61d, 1970), an event which normally occurs at the time of initiation of DNA replication. Late mRNA has been divided into two classes: (1) true-late, synthesized only from newly replicated DNA and only in the presence of gene 55 product, and (2) quasi-late, synthesized in limited amounts from parental DNA in the absence of gene 55 product (Salser et al., 1970; Bruner and Cape, 1970). The gene 55 product appears to be a simple, continuously acting, phage specific positive control element (Pulitzer, 1970; Pulitzer and Geiduschek, 1970). Introduction of a gene 55 mutation eliminates the early low rate of DNA synthesis, suggesting that the gene 55 product temporally regulates the onset of the second, rapid DNA synthesis mode (see below). The DNA synthesized by the first, slow DNA synthesis mode may be the DNA competent for late transcription. The functions of genes 39, 41, 44, 45, 52, 59, 60, 61, and 62 remain unknown. A recent study of DNA delay mutants, DD mutants (genes 39, 52, 60, and 61), indicates that initiation of DNA synthesis occurs at the normal time in DD mutants, but that the initial rate of synthesis is slower (Yegian et al., 1971). Later in infection, the rate of synthesis abruptly increases, suggesting two distinct modes of synthesis. This abrupt change does not occur during chloramphenicol treatment, suggesting that a protein must be synthesized to effect the change (Bolle et al., 1968a, b; Yegian et al., 1971). Consistent with these observations, Frankel et al. (1968) observed that a membrane-bound T4 DNA polymerase is present early in infection, but is replaced late in infection by a new DNA polymerase activity, found in the supernatant of cell extracts. Altman and Lerman (1970a) have presented evidence for two stages in T4 DNA replication. In the first stage, a protein-DNA--cell membrane complex is formed early in infection, and in the second stage, occurring late in infection, a rapidly sedimenting DNA species is found whose properties suggest the presence of branches or loops (concatemeric DNA). This latter stage may be related to the DNA synthesis modes observed by Delius et al. (1971), illustrated in Fig. 23A, and by Werner (1968). 2-aminoacridine appears to block release of parental DNA from the replicative complex (Altman and Lerman, 1970b). Scotti (1969) has determined the number of viable phage in thermosensitive T4 mutants as a function of time after infection of shift to the restrictive temperature. His data suggests that the products of genes 42, 44, and 45 act both as controlling elements and as enzymes, and that the gene 30 and gene 41 products act in a cooperative manner. Oishi (1968c) has shown that newly synthesized DNA accumulates as single-stranded DNA in gene 41 mutants, concluding that the gene 41 product is necessary to convert the single-stranded DNA into a double-stranded species with nicks or short gaps. (b) T7. The genome of bacteriophage T7 is a single double-stranded DNA molecule of molecular weight 20 x l 0 6 daltons (Richardson, 1966), with information for about 30 genes (Studier and Maizel, 1969). Nineteen essential genes, identified via isolation of conditionally lethal mutants, have been reported (Hausmann and Gomez, 1967; Studier, 1969; Studier and Hausmann, 1969). Genes 1 through 6 are necessary for DNA synthesis (Hausmann and Gomez, 1967; Hausmann and LaRue, 1969; Studier, 1969). T7 endonuclease I is the product of gene 3 (Sadowski, 1971; Center and Richardson, 1970a, b; Center et al., 1970) and an exonuclease is the product of gene 6 (Kerr and Sadowski, 1972a, b). The product of gene 5 is a DNA polymerase (Grippo and Richardson, 1971). The gene 1 product acts as at positive control element for most of T7 gene expression (Studier and Maizel, 1969; Siegel and Summers, 1970). Gene 1 mutants do not yield viable phage or lyse the host cell (Haus-

356

DOUGLAS W. SMITH

mann and LaRue, 1969; Studier, 1969), and make very few RNA or protein species (Siegel and Summers, 1970; Studier and Maizel, 1969). Initially thought to be a sigma-like factor (Burgess et al., 1969; Travers and Burgess, 1969) active in a complex with host RNA polymerase (Summers and Siegel, 1970), the gene 1 product has now been shown to be a unique T7-induced RNA polymerase (Chamberlin et al., 1970; Summers and Siegel, 1970a, b; Chamberlin and McGrath, 1970). Studier (1969) and Hausmann and Gomez (1967) have shown that gene 1, 4 and 5 mutants exhibit no detectable DNA synthesis, whereas gene 2, 3 and 6 mutants synthesize some DNA but stop synthesis prematurely (DA phenotype). Host DNA breakdown is blocked in gene 1 mutants, indicating that host DNA breakdown is a late function. Gene 3 and 6 mutants also prevent host DNA degradation (Center et al., 1970a, b); their products participate directly in this degradation (Kerr and Sadowski, 1972b). Gene 8, 9, 18, and 19 mutants lead to the accumulation of rapidly sedimenting DNA species (Hausmann and LaRue, 1969). Phage T7 induces a new DNA ligase upon infection (Becker et aL, 1967) which is apparently not the product of any of the 19 known complementation groups. Recently, a T7 amber mutant deficient in this ligase has been isolated (Masamune et al., 1971b). In either an su ÷ or s u - host, the DNA synthesized has normal sedimentation characteristics and the UV-sensitivity of the phage is normal. However, in a ligase-deficient E. coli mutant, N1318 (Gellert and Bullock, 1970), small DNA fragments (1 IS) accumulate during infection with the T7 ligase mutant, suggesting that the T7 ligase is involved in T7 DNA replication, but that the host ligase can substitute when necessary. The ligase mutation has not been mapped. (c) Lambda. The genetics and physiology of lambda development has been exhaustively studied and reviewed (Dove, 1968; Echols and Joyner, 1968; Radding, 1969; Calendar, 1970; Hershey, 1971); we consider only specific relevant aspects of this area. The lambda chromosome is a double-stranded DNA molecule of molecular weight 32 × 106 daltons. It contains approximately 50 genes of which about 38 genes have been identified (Fig. 13). These known genes, identified primarily via isolation of conditionally lethal mutants (Campbell, 1961; Parkinson, 1968), are clustered according to function (Fig. 13). Genes involved in recombination, either in the specialized recombination events leading to integration and excision, or in the generalized phage-specific recombination events, are found near the center of the vegetative lambda map (Hershey, 1971). Genes O and P are the only genes yet discovered which are specifically required for DNA replication (Brooks, 1965; Eisen et al., 1966; Joyner et aL, 1966; Ogawa and Tomizawa, 1968; Rambach and Brachet, 1971). These genes may control synthesis of an endonuclease (Shuster and Weissbach, 1968a, b, 1969; Freifelder and Kirschner, 1971). The replication origin has been located in the right half of the vegetative chromosome by physical studies (Makover, 1968a; LePecq and Baldwin, 1968; Schn6s and Inman, 1970), and between the C~ and O genes by genetic means (Eisen et al., 1966; Dove et aL, 1969). Two mutants, t5 and tl 1, are cis-dominant in DNA replication, i.e., these mutants do not replicate even when the required gene products are provided in trans. Recently, 40 more cisdominant DNA replication-defective mutants have been isolated, of which one, til2, has been studied extensively (Dove et aL, 1971). The mutant t5 is thought to be a deletion mutant of the replication origin and part of gene 0; ti 12 maps near t5 to the left of all O gene mutants, and may be a point mutation (Dove et al., 1971). In addition to its role in the control of transcription, the CI gene product, which is the

(b)

FUNCTIONS :

T 2 7 ~2

II

CI

X

2'0

Ii

I M M 21

Y CflRl 0 c-,~ I , I, ,I ' t 2 (Ind-) Cp ,I f i l 2 V~Vs , i , . , ~ . , t 5 a I M M 4.'.34 T. ORIGIN

REX

(3 uJ "1-

i

d

37% b2 2'5

~ ~

1434

i21

--

-

z

rr~ ~OX ~_w

-

0::

L)

-,

),dv

EARLY CONTROLS

CL LU n-"

Co ~E 0

Z Ic~[ 0 "<"3 CL Lu (~

(/) W fY

~

Z

<_o

o

--

g

(A d 09

SR

'~Z -JO O

o

IM M U N I T Y REGION 4:5% 485% aa'intxisexo/~'cmNCTxycgOPAbl" o L 5'0 I 3'5 i 4"0 ( 4 5 ~ Red'-

"ru~ 0... a OZ n- w a_ 5' 4 8 5'--A

42%

FIG. 13. Genetic and D N A map of bacteriophage lambda, A. Genetic and D N A map of vegetative lambda. B. Detailed enlargement of the "early control" region of the lambda map. Adapted from Szybalski et al. (1970), Dove (1968), and Dove et al. (1971).

I

C111" N

"J I-

% ( G+C): 48% 57% GENES: 5'G AWB C DEFZUVGTH MLKI NUCLEOTIDE : 0 l 5 I I'0 I 1"5 P A I R S Y I0 -z' :3' ~ . . . . . w.--,,,,~-

(a)

DNA STRAND: L r

"-4

t~

?.

o~ ,0

358

DOUGLAS W. SMITH

lambda repressor (Ptashne, 1967), also appears to directly inhibit DNA replication: superinfection of a lysogenic cell containing a replicating lambdalmm434 helper phage does not lead to replication of the superinfecting phage (Thomas and Bertani, 1964). Mutants constitutive for D N A replication (riO-constitutive for the replication-inhibition site) have been isolated; these map in the same region as the replication origin (Dove et al., 1969). The ri¢ mutants synthesize the "O-P messenger" constitutively (Dove et al., 1969, 1971), whereas t l l does not synthesize this messenger normally (Cohen and Hurwitz, 1968; Skalka et aL, 1967; Taylor et al., 1967). This suggests that synthesis of the O-P messenger may be necessary for DNA replication, and replication control by repressor is only an indirect consequence of this transcription event. Another alternative to explain replication inhibition would invoke a cis-acting gene product (see below). However, such has not been found in the phage lambda system, and genetic data argue strongly against this possibility (Dove et al., 1969, 1971). (d) P2. P2 is a noninducible, temperate bacteriophage (Bertani, 1951; Bertani and Bertani, 1970) containing a double-stranded DNA chromosome of molecular weight 22 x 106 daltons (Mandel, 1967; Mandel and Berg, 1969; Inman and Bertani, 1969). Eighteen essential phage genes, of a potential 35, have been identified; these are clustered with regard to function (Bertani, 1968; Calendar, 1970). Gene A of P2 appears to exert positive control over the expression of all P2 genes by way ofa cis-acting protein (Lindahl, 1969a, b; 1970). A cis-acting protein is defined to be one that functions only on the chromosome containing the structural gene for the protein. Gene A mutants do not synthesize DNA, do not kill the host, and cannot be complemented. Gene B mutants also do not synthesize DNA or kill the host. In high ionic strength media, complementation between gene A and gene B mutants occurs, with a burst of replicated B mutant phage and of unreplicated A mutant phage. It is thought that replication of the A mutant DNA does not occur, even in the presence of replicating B mutant DNA, because the A gene product is required and can act only in cis, i.e., only on the DNA molecule leading to its production. It is thought the P2 repressor, the gene C product, simply prevents expression of the required gene A c/s-acting protein, although a more complex explanation involving a transcription event has not been ruled out. Integrative suppression by P2 sig mutants as prophages on the thermosensitive dna mutant, dnaA46, was discussed above (section IIIB2). (e) ff x 174 and S13. The biology of these extensively studied bacteriophages has been thoroughly reviewed (Hoffmann-Berling et al., 1966; Sinsheimer et aL, 1967; Sinsheimer, 1968a, b; Ray, 1968; SinsheimeretaL, 1968; Pratt, 1969; Calendar, 1970). q~x 174 and S13 are two closely related small spherical DNA bacteriophages, having as their genome a singlestranded circular DNA molecule of molecular weight 1.7 x 106 daltons (Sinsheimer, 1959a, b). The corresponding double-stranded DNA molecule, of molecular weight 3.4 x 106 daltons, contains information for about 5 average size genes. Eight, and possibly ten, cistrons have been discovered; their designations and functions are given in Table 2. Upon infection, the single-stranded DNA is rapidly converted into a double-stranded molecule (Sinsheimer et al., 1962; Tessman, 1966), the parental replicative form (RF). Only host enzymes are needed for parental RF formation. The parental RF becomes associated with an intracellular "site" (Yarus and Sinsheimer, 1967; Knippers and Sinsheimer, 1968a). RF replication requires the cistron A product (Tessman, 1966; Lindqvist and Sinsheimer, 1967; Sinsheimer, 1968a). This protein product is synthesized in the presence of 30 tzg/ml chloramphenicol (CM-resistant protein) and has been isolated using a double-label method (Levine and Sinsheimer, 1969a). Since single-strand (SS) DNA synthesis will not proceed in

359

D N A SYNTHESISIN PROKARYOTES"REPLICATION TABLE 2 CISTRONS OF PHAGE 6X 174 AND S13

Cistron designation ~b× 174 Sinsheimer Vl IV VIII V I

VII III II

~b× 174 Hayashi C B

H(?) D G E F A (I)

O)

S13 Tessman IV II VI(?) VII V I IIIa IIIb

New A B

C D E F G H (I) (J)

Function RF replication Spike component (SS synthesis) SS synthesis SS synthesis Lysis Major capsid protein (SS synthesis) Spike body (SS synthesis) Spike tip (lysis)

RF: replicative form DNA. SS: single-stranded DNA. These genetic results are well summarized by Sinsheimer et al. (1967), Tessman (1967), and Jeng and Hayashi (1970).

the presence of 30/~g/ml CM, it has been possible to study RF replication alone. Further, Lindqvist and Sinsheimer (1966) observed that in an h c r - host, pretreatment of the host with mitomycin C prevented host D N A synthesis, yet permitted phage D N A synthesis in a presumed normal manner. R F replication proceeds in a semiconservative manner (Denhardt and Sinsheimer, 1965) only at the cell site. The replicating R F is an RFII species (Francke and Ray, 1971), and the parental D N A strand remains attached to the site throughout infection. The progeny RF molecules do not appear to be attached to a cell site, and both form I (RFI) and form II (RFII) progeny D N A species are found in lysates. At about 12 min after infection at 37 °, host D N A synthesis and net phage RF synthesis cease (Lindqvist and Sinsheimer, 1967) and SS D N A synthesis commences, at a rate 5 to 10 times that of RF synthesis. SS D N A synthesis requires the cistron C and cistron D products; these products are not found as components of the mature phage particles (Sinsheimer et al., 1967; Tessman, 1967; Jeng and Hayashi, 1970; Funk and Sinsheimer, 1970). Further, the products of cistrons B, F, and G are needed for SS synthesis, although these products are phage particle components (Dowell and Sinsheimer, 1966; Knippers and Sinsheimer, 1968b; Siegel and Hayashi, 1969; Burgess and Denhardt, 1969; Iwaya and Denhardt, 1971). Since singlestranded ~ x 174 D N A is not found free in an intracellular pool (Sinsheimer et al., 1962; Knippers and Sinsheimer, 1968b), the phage particle components probably act in a positive manner to promote SS D N A synthesis or to stabilize the synthesized SS DNA. Cistrons E and H are not needed for either RF or SS D N A synthesis, and both are needed for cell lysis. The cistron H product is found as a component of the phage particle coat. I and J are recent discoveries. ( f ) f d and M13. The physiology and genetics of the small filamentous D N A phages have been recently reviewed (Ray, 1968; Pratt, 1969; Marvin and Hohn, 1969; Calendar, 1970). MI3 and fd, two examples of these bacteriophages, have a single-stranded circular D N A molecule of molecular weight about 2 × 106 daltons as their genome (Marvin and Hohn,

360

DOUGLAS W. SMITH 3'

P

Template

'~' PrimeJ B

r_ P

T

T

T 3' P

-P T

T

0 ligodeoxynuc leo?ide primer D

i

,

r

i

l i l t

I

~

~

I~

~

I

~

r

~

r

I

r

~

~

,

,

i

I

r

i

I

d

3'

FIG. 14. Schematic representation of the principle activities of E. coli DNA polymerase I. A. Repair synthesis of partially single-stranded DNA substrate, showing the primer (P) and template (T) parts of the DNA substrate. B. Repair synthesis of a gapped region in a double-stranded DNA substrate. C. Conversion of a single-stranded circular DNA substrate into a double-stranded species, showing the requirement for an oligodeoxynucleotide as primer (Goulian, 1968a, b). D. Nick translation. Combined 5"- to 3"-exonucleaseand polymerase activities. E. Possible mechanism for extensive synthesis at temperatures above 25°C as suggested by Mitra and Kornberg (1966), leading to a branched DNA product. Adapted from Richardson (1969), Kornberg (1969), and Goulian (1971). 1969). Genetic analysis of conditionally lethal mutants has shown the presence of eight cistrons (Pratt et al., 1966, 1968, 1969). The sequence of events following infection are rather similar to those following infection with phage ff × 174. The parental single-stranded D N A is first converted into an RF form, the parental R F replicates semi-conservatively, and at about 10 min after infection, SS D N A synthesis begins. The products of genes 1 (unknown product), 3 (adsorption protein A), 4 (unknown), 6 (unknown), and 8 (major coat protein B) are not required for either R F D N A synthesis or SS D N A synthesis. The gene 2 product is necessary for progeny R F synthesis, but not for the parental R F formation. The gene 5 product is required for SS D N A synthesis (Forsheit et al., 1971; Salstrom and Pratt, 1971), and has recently been isolated and shown to be a renaturation-denaturation protein (Alberts et aL, 1971). The gene 7 product has not been classified. Distinctions between the filamentous and spherical phages include the following: the filamentous phages (1) accumulate a pool of SS D N A late in infection, (2) do not

D N A SYNTHESIS IN PROKARYOTE$"REPLICATION

361

require structural phage coat proteins for SS DNA synthesis, and (3) do not lyse the host cell, but rather extrude phage into the growth medium. C. Enzymes and Other Proteins 1. D N A polymerases (a) E. coli D N A polymerase L DNA polymerizing activity was first detected in Escherichia coli in 1958 (Bessman et al., 1958), and the subsequent isolation and study of the enzyme has been extensively and periodically reviewed (Kornberg, 1961; Bessman, 1963; Bollum, 1963a; Richardson et al., 1963a, Mitra and Kornberg, 1966; Englund et aL, 1968; Richardson, 1969; Kornberg, 1969; Goulian, 1971). A modified isolation procedure (Jovin et al., 1969a) resulted in the isolation of 0.6 g of purified enzyme from 90 kg of E. coli B. This permitted extensive studies of the physicochemical properties, binding sites, and active center of the enzyme, summarized below. Physicochemical properties and binding sites. The enzyme has a molecular weight of 109,000 daltons (Jovin et al., 1969a), and contains 2 to 4 g atoms Zn ÷ ÷ per mole enzyme (Slater et al., 1971). In spite of its high molecular weight, it appears to be a single polypeptide chain (Englund et al., 1968; Jovin et aL, 1969a), as shown by the following criteria. First, the enzyme molecular weight, determined by equilibrium sedimentation, is the same after denaturation in 6 M guanidinium-C1 as before. Second, during electrophoresis in SDS-acrylamide gels, the enzyme migrates as a single polypeptide species of molecular weight 105,000 daltons at pH 3.5, 8 and 11. Third, amino acid analysis shows a single sulfhydryl group and single disulfide group per 109,000 daltons, and 0.6 mole N-terminal methionine per 109,000 daltons. Finally, during renaturation of a denatured preparation, both polymerase and exonuclease II activities are recovered in a concentration independent manner. However, the amino acid sequence is as yet unknown, and the possibility of a nonpeptide linker still exists. DNA binding studies using a variety of radioactive DNA substrates and Hg2°a-labeled enzyme (Jovin et al., 1969b) have demonstrated that DNA polymerase I binds in a Mg ÷ ÷independent reaction to the ends or nicks in helical double-stranded DNA, but binds to single-stranded DNA at multiple sites at a density of about one enzyme molecule per 220 nucleotides (Englund et al., 1969b; Kornberg, 1969). Electron microscopy has been used to observe the binding to a nicked synthetic DNA substrate (Griffith et aL, 1971). Using 20 microliter capacity chambers for equilibrium dialysis studies of deoxynucleoside-5'-triphosphate (dNTP) binding to polymerase, only one dNTP binding site per enzyme molecule was found (Englund et al., 1969a). Competition experiments demonstrated a requirement for three phosphate groups on the substrate molecule, but little sugar or base specificity, although purine substrates bind better than do pyrimidine substrates. Effects of a concomitant DNA substrate on the dNTP binding were not examined in these studies. However, a recent study of dNTP binding to a DNA polymerase-DNA complex using a partially purified rat hepatoma DNA polymerase indicates a specificity for the entire dNTP molecule (Ove and Laszlo, 1969). Deoxynucleoside-5'-monophosphates (dNMPs), deoxynucleoside-5'-diphosphates (dNDPs), deoxynucleosides, and pyrophosphate bind to DNA polymerase ! in a site which is probably the primer terminus site (Fig. 15). Studies with a series of analogues have shown a requirement for a 3'-hydroxyl (3'-OH) group in the "ribo" configuration in the ribose moiety (Atkinson et al., 1969). dNTPs do not compete for this site, indicating that it is not

362

DOUGLAS W. SMITH

the triphosphate binding site (Huberman and Kornberg, 1970). Recent electron spin resonance studies using spin-labeled substrate analogues indicate that the triphosphate and primer terminus sites (Fig. 15) are only about 7 A apart on the polymerase molecule (Krugh, 1971). Enzymatic actioities and the active center. Five distinct enzymatic activities are associated with the purified D N A polymerase I. These consist of the following: (1) polymerization (5'- to 3'-chain growth); (2) 3'-hydroxyl to 5'-hydrolysis (exonuclease activity), with release of 5'-dNMPs; (3) 5'-phosphate to 3'-hydrolysis (exonuclease activity), with release of 5'dNMPs, and dinucleotides or oligonucleotides (4) 3'-hydroxyl to 5'-pyrophosphorolysis, utilizing pyrophosphate to liberate 5'-dNTPs, and (5) pyrophosphate-triphosphate exchange.

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FIG. 15. The active center of Escherichia co# DNA polymerase I. A. The three binding sites for the DNA substrate molecule and the one deoxynucleoside triphosphate binding site. B. The catalytic activities of the enzyme: (1) polymerization; (2) 3'- to 5'-exonucleolytic hydrolysis; (3) 3"- to 5'-exonucleolytic pyrophosphorolysis; (4) 5'- to 3"-exonucleolytic hydrolysis; (5) triphosphate exchange is thought to be a combination of polymerization and pyrophosphorolysis. Adapted from Kornberg (1969). D N A polymerase I will bind to multiple sites on single-stranded D N A or to nicks and ends of double-stranded DNA. However, the polymerization reaction will proceed only if the D N A substrate contains a template strand, a primer strand, and a 3'-OH primer terminus (Figs. 14 and 15). These portions of the D N A substrate are thought to occupy three sites in the polymerase "active center", the template site, the primer site, and the primer terminus site, the site which binds 5'-dNMPs. Two other sites are present in the active center: (1) the triphosphate binding site, and (2) a site for the 5'-phosphate terminus of a nicked D N A substrate (see Fig. 15). In addition to the appropriate D N A substrate, the four 5'-dNTPs and Mg ÷ ÷ are required for polymerization (Kornberg, 1961; Sheu et al., 1970). The triphosphates of some base analogues will substitute for the four common bases (Kornberg, 1961; Bessman, 1963;

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Roy-Burman et al., 1970; Lezius, 1971). Further, the purified enzyme can apparently use 5'-dNDPs when another nucleoside triphosphate is present to serve as a phosphate donor. This suggests the presence of a nucleoside diphosphokinase activity associated with the DNA polymerase (Miller and Wells, 1971). An ideal DNA substrate for DNA polymerase I is an exonuclease III-digested doublestranded DNA molecule (Fig. 14A). Exonuclease III, a combined phosphatase-exonuclease, catalyzes hydrolysis of a double-stranded DNA molecule from the two 3'-ends, releasing 5'-dNMPs and yielding a partially double-stranded DNA molecule (see section IIIC 3). The polymerization reaction of DNA polymerase I proceeds via a nucleophilic attack of the 3'-OH-group of the DNA primer terminus on the s-phosphate of the appropriate dNTP bound to the triphosphate site of the enzyme (Fig. 15B), with release of pyrophosphate (Kornberg, 1961). The rate of synthesis is high and accurate; this reconversion of the partially single-stranded DNA substrate into a double-stranded DNA species (Fig. 14B) has been termed the repair reaction (Richardson et al., 1964a). DNA polymerase I can use denatured DNA or partially single-stranded DNA as a substrate. With linear single-stranded DNA, presumably the 3'-OH-terminus loops back to provide a DNA primer (Bollum, 1967; Goulian et al., 1968). Part of the 3'-OH-end may first be degraded by the 3'-to 5'-nuclease, before polymerization begins, as is observed using the T4 DNA polymerase (Englund, 1971). The product DNA is found covalently linked to the DNA substrate (Bollum, 1963b; Schildkraut et al., 1964; Goulian et al., 1968; Steuart et al., 1968). Circular single-stranded DNA (Fig. 14C) will serve as an adequate DNA substrate for E. coli DNA polymerase I only if an oligodeoxynucleotide "primer" is added (Goulian and Kornberg, 1967; Goulian, 1968a, b). This oligomer is covalently bound to the newly synthesized DNA. The phage T5 DNA polymerase and various animal cell DNA polymerases are also able to use such oligomer primers (Smellie, 1963; Keir, 1965; Bollum, 1967), but the T4 phage DNA polymerase apparently cannot. The product DNA can be freed from the template DNA by denaturation (Mitra et al., 1967; Goulian and Kornberg, 1967), yielding the circular template strand and the linear primer-product strand. The product has the same molecular weight as the template, and the fidelity of using the circular strand as template is sufficiently high that the end product is biologically active (Goulian et aL, 1967) in the ~ × 174 spheroplast assay system (Guthrie and Sinsheimer, 1963). When double-stranded DNA is used as a substrate for the DNA polymerase I, the rate of synthesis is low (Richardson et al., 1963a; Richardson et al., 1964a). The product DNA renatures with unusually high kinetics during cooling after heat-denaturation (Schildkraut et al., 1964), contains branches as seen in the electron microscope (Inman et al., 1965), and is biologically inactive (Richardson et al., 1963b). Models (Fig. 14E) have been proposed to account for branching and nondenaturability in which polymerase either reverses its direction of synthesis in a hairpin loop, using the product DNA as a template, or shifts from using one of the substrate DNA strands as template to using the other displaced strand as template (Schildkraut et al., 1964; Mitra and Kornberg, 1966; Bollum, 1967). This in vitro synthesis, known and defined as "extensive synthesis", does not occur below 22 °, is favored by low salt, and yields a product several times the length of the template DNA (Mitra and Kornberg, 1966; Richardson, 1969; Goulian, 1971). With single-stranded circular DNA, synthesis by DNA polymerase I following completion of the repair reaction is of this extensive synthesis type (Mitra et al., 1967). The 3'- to 5'-hydrolysis reaction of E. coli DNA polymerase I, found in the initial studies

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DOUGLAS W. SMITH

of the enzyme (Bessman et aL, 1958), was termed exonuclease II (Lehman and Richardson, 1964). The 5'- to 3'-hydrolysis was found only recently (Klett et al., 1968) and was termed exonuclease VL However, since both activities appear to be associated with the same enzyme (indeed, with the same polypeptide chain), these will be termed the 3'- to 5'-exonuclease II and 5'- to 3'-exonuclease II activities of E. coli DNA polymerase I. The 3'- to 5'-nuclease hydrolyses single-stranded DNA better than double-stranded DNA (Cozzarelli et aL, 1969; Kelly et al., 1970), requires a 3'-OH group, and releases 5'-dNMPs (Lehman and Richardson, 1964). Mismatched deoxynucleotides can be removed from the 3'-primer terminus by the 3'- to 5'-nuclease activity (Brutlag, 1971). In contrast, the 5'- to 3'-nuclease requires a double-stranded substrate (Cozzarelli et al., 1969) and will hydrolyse DNA containing either a 5'-OH group or a 5'-phosphoryl group (Kelly et al., 1969). About 25~o of the hydrolysis products are dinucleotides or longer, with low pH favoring production of oligonucleotides (Deutscher and Kornberg, 1969b; Kelly et al., 1969; Kadohama and McCarter, 1970). A polydeoxyribonucleotide substrate with a 5'-triphosphate is hydrolysed, releasing a dinucleoside tetraphosphate. This suggests that the triphosphate is bound to the polymerase triphosphate binding site, and that the site for 5'- to 3'-hydrolysis is adjacent to this site (Kornberg, 1969). The 5'- to 3'-nuclease activity of DNA polymerase I is stimulated by concurrent synthesis (observed by adding the four dNTP's) using a nicked DNA substrate (Kelly et al., 1970). A mechanism involving concurrent synthesis and hydrolysis, at equivalent rates and probably by the same enzyme molecule, has been proposed and termed "nick translation" (Fig. 14D). This reaction is most easily observed using a synthetic polydeoxyribonucleotide, poly(dA)oligo (dT), as substrate. With nicked DNA, this reaction is obscured by the "extensive synthesis" reaction discussed above (Masamune and Richardson, 1971). Pyrophosphorolysis, the reverse of the polymerization reaction, is the pyrophosphate mediated cleavage of DNA by E. coli DNA polymerase I with liberation of dNTPs (Richardson et aL, 1964b; Deutscher and Kornberg, 1969a). Pyrophosphate exchange is a reaction similar to the combined actions of polymerization and pyrophosphorolysis, resulting in exchange of the pyrophosphate moieties of the dNTPs. Presumably, a covalent bond is formed between the 3'-OH group of the DNA primer and the a-phosphate of the incoming dNTP, prior to pyrophosphorolysis. However, pyrophosphorolysis, for unexplained reasons, appears to proceed more slowly than pyrophosphate exchange, and is inhibited by the dNTPs (Kornberg, 1969). Since pyrophosphate exchange proceeds during synthesis by the enzyme (Deutscher and Kornberg, 1969a), p32-pyrophosphate can be used in a convenient assay for DNA polymerase I. Separation of the enzymatic activities. As mentioned above, the five enzymatic activities of DNA polymerase I appear to be associated with a single polypeptide chain. However, acylation of the enzyme with N-carboxymethylisatoic anhydride results in a differential loss of the polymerization activity relative to the 3'- to 5'-nuclease activity. For example, at pH 7.4, the derivative possesses only 0.2~o of polymerization activity but 920~ of the 3'- to 5'-nuclease activity (Jovin et al., 1969b). Similarly, differential recoveries of activities following denaturation are observed (Jovin et aL, 1969a). Further, limited digestion of the 109,000 dalton enzyme with subtilisin or trypsin results in two fragments, one of molecular weight about 75,000 daltons, and the other about 35,000 daltons (Brutlag et al., 1969; Klenow and Overgaard-Hansen, 1970; Klenow and Henningsen, 1970; Klenow et aL, 1971). The larger fragment retains the polymerization, pyrophosphorolysis, and 3'- to 5'-nuclease activities, but not the 5'- to 3'-nuclease activity. The smaller fragment lacks any enzymatic activity,

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unless proteolytic cleavage is permitted in the presence of DNA; it then exhibits 5'- to 3'nuclease activity (Klenow and Overgaard-Hansen, 1970). Other low molecular weight DNA polymerase species have been observed (Hori et al., 1966; Lezius et al., 1967; Cavalieri and Carroll, 1968; Yoshida and Cavalieri, 1970, 1971). Whether these species are biologically significant or proteolytic artifacts remains to be definitively determined. (b) E. coli D N A polymerase II. The isolation of mutants of E. coli deficient in DNA polymerase I (DeLucia and Cairns, 1969) which are not conditionally lethal has prompted the search for further DNA polymerizing activities in these cells. A second activity, now called DNA polymerase II, has been studied by four groups. Kornberg and Gefter (1970), using severe lysis via a French pressure cell of E. coli polA1 and polA4 mutants, partially purified (about thirty-fold) a polymerizing activity on high salt (500 m i KC1) and low salt (50 mM KCl) glycerol gradients. Further purification to about 4000-fold enrichment (Kornberg and Gefter, 1971), using DEAE and phosphocellulose chromatography, has yielded a homogeneous preparation as determined by gel electrophoresis. Knippers (1970) used Triton XI00 to solubilize an activity from a membrane DNA synthesis system (Knippers and Str~itling, 1970) prepared from gently lysed E. coli W3110 polA1 endoIcells. A similar activity could be found in the membrane fractions obtained from polA ÷ if these fractions were initially washed extensively. This activity was estimated to comprise 1 to 29/o of the total E. colipolA ÷ polymerizing activity. Using DEAE and phosphocellulose purification of toluene-treated (Moses and Richardson, 1970a) E. coli D110 polA1 endoIcells (Moses and Richardson, 1970b) or E. coli W3110 polA ÷ or polA÷dnaB mutants (Moses and Richardson, 1970c), a polymerizing activity called DNA polymerase II has been purified to homogeneity (about 3000-fold enrichment) as determined by polyacrylamide gel electrophoresis. In polA ÷ cells, 5 to 109/o of the total DNA polymerase activity was estimated to be due to DNA polymerase II. Wickner et al. (1972a) used alumina grinding and a phase partition system, in addition to chromatography, to purify DNA polymerase II 27,000-fold and in high yield from E. colipolA-endoI- cells and E. colipolA + minicells. In all cases, the isolated DNA polymerase II requires a nicked or sonicated doublestranded DNA substrate, all four dNTPs, Mg + +, and is not stimulated by ATP. The enzyme is sensitive to high salt (0.2 M K C D and to sulfhydryl agents, e.g. pCMB, but is insensitive to anti-DNA polymerase I antibody and is unable to use poly d(A-T) as a primer-template substrate. Each of these properties distinguish DNA polymerase II from DNA polymerase I. Kornberg and Gefter (1971) found a preference of the enzyme for partially single-stranded DNA (exonuclease III-treated DNA). The solubilized enzyme (Knippers, 1970; Moses and Richardson, 1970a; Wickner et al., 1972a) preferred either exonuclease III-treated DNA as substrate, exhibiting lower activity with nicked or sonicated DNA. DNA polymerase II is thought to require a duplex DNA substrate with a single-stranded region having 3'-OH primer terminus groups. The DNA product is double-stranded DNA, with product covalently attached to the primer DNA (Wickner et al., 1972b). DNA polymerase II thus catalyses only a repair reaction, with no extensive synthesis. DNA polymerase II isolated from the dnaB mutant cells exhibits no thermosensitivity (Moses and Richardson, 1970c). Loeb et al. (1971) have also shown that polymerizing activity can be detected in E. coli polA + cell extracts which is insensitive to antiserum against DNA polymerase I, relatively insensitive to ethanol, and sensitive to high salt. The isolated DNApolymerase II can catalyse both pyrophosphorolysis and a phosphate exchange reaction. It also has a 3'- to 5'-exonuclease activity against a single-stranded DNA substrate, but no 5'- to 3'-nuclease activity has been detected. Gel filtration suggests a

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DOUOLASW. SMITH

molecular weight of about 120,000, and there are estimated to be about 17 enzyme molecules per bacterium (Wickner et al., 1972a). The cytidine analogue, 1-fl-D-arabinofuranosylcytosine (ara-C), is a specific inhibitor of DNA synthesis in bacteria (Cohen, 1966), and the effects of the 5'-triphosphate of ara-C, ara-CTP, on several in vitro systems and on isolated E. coli DNA polymerases I, II, and III and the DNA polymerase induced by phage T4 have been examined (Rama Reddy et al., 1971). Both in toluene-treated E. colipolAl andpolA + cells and in a lysed cell system derived from E. coli W3110 polA I, ara-CTP specifically inhibits the ATP-stimulated DNA synthesis, having relatively little effect on the ATP-independent synthesis. Polymerase I was insensitive to ara-CTP, polymerase III was inhibited only at high ara-CTP concentrations, and both polymerase II and T4 DNA polymerase were inhibited about 50~o at the lowest ara-CTP concentration yielding maximal inhibition in the in vitro systems. These data were interpreted to mean that polymerase II is likely to be a replicative DNA polymerase, or replicase. (c) E. coil D N A polymerase III. In their purification of DNA polymerase II on a phosphocellulose column, Kornberg and Gefter (1971) noted the presence of two polymerase peaks, which they called A and B. The two activities were different in several respects: (1) B was stimulated about two-fold by 10 mM (NH4)2SO4, whereas A was inhibited by all concentrations of (NH4)2SO4; (2) A is about three-fold more sensitive to both NEM and to heat inactivation than is B; and (3) A is less stable during storage than is B. It was not possible to interconvert A and B. A and B appear to have the same reaction requirements, and both are insensitive to antiserum against DNA polymerase I. Two recent independent investigations have shown that DNA polymerase III (peak A of Kornberg and Gefter, 1971) is the gene product of certain dnaE thermosensitive mutants. Gefter et al. (1971) have constructed a series of E. coli dna(A-G) double mutants containing the polA1 mutation and one of the thermosensitive dna mutants (see section IIIB). These were each tested for the presence of the A and B peaks at 45 ° and 30 °. All mutants tested contained wild-type activities of peak B, now called DNA polymerase II, at the two temperatures. However, the ratio of activity at 45 ° to that at 30 ° for peak A, now called DNA polymerase III, was greatly reduced in two of four dnaE mutant strains tested; the ratio in the fifth strain was reduced somewhat. All other mutants exhibited wild-type ratios, as did dna + revertants of the dnaE mutants. Thus, DNApolymerase II and DNA polymerase III are distinct enzymes, and DNA polymerase III, the first enzymatic activity to be identified with a thermosensitive DNA replication mutant, seems to be required for DNA replication in E. coll. Niisslein et al. (1971)have isolated a series of thermosensitive DNA replication mutants and tested these in their cellophane in vitro DNA synthesis system (Schaller et al., 1972). Four mutants containing a thermosensitive soluble factor do not complement each other in this in vitro system, and map in the dnaE region (see Fig. 7). Using complementation in the cellophane DNA system prepared from these mutants, the factor has been purified 1000fold, and shown to have properties similar to DNA polymerase III. It is particularly interesting that, in a standard DNA polymerase assay using exonuclease III-treated calf thymus DNA as substrate, the isolated enzyme exhibits the salt sensitivity and relative low rate of synthesis in low salt observed by others (see above). However, when used at high concentration on the cellophane or when used to complement the cellophane DNA system prepared from dnaE mutants, the salt sensitivity disappears, and the specific activity increases about five-fold.

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(d) Other DNA polymerases. DNA polymerase enzymes have been extensively purified from Bacillus aubtiIis (Okazaki and Kornberg, 1964), Micrococcus luteus (Zimmerman, 1966; Litman, 1968; Harwood et al., 1970a, b; Harwood and Wells, 1970), Mycobacterium smegmatis (McNulty and Winder, 1971), from E. coil infected with bacteriophages T2 (Aposhian and Kornberg, 1962), T4 (Goulian et al., 1968), T5 (Orr et al., 1965; Steuart et al., 1968), and T7 (Oey et al., 1971 ; Grippo and Richardson, 1971), and from calf thymus (Yoneda and Bollum, 1965; Bollum, 1963b) and other eukaryotic sources. The bacterial DNA polymerases in general possess the 5'- to 3'-nuclease activity, can utilize doublestranded DNA substrates, and can execute the extensive synthesis reaction. In contrast, phage enzymes lack the 5'- to 3'-nuclease activity but contain a 3'- to 5'-nuclease activity, require a single-stranded DNA substrate, and do not exhibit the extensive synthesis reaction. As with E. coli DNA polymerase I, the nuclease and polymerase activities of T4 DNA polymerase are separable. The 90,000 dalton fragment of the T4 enzyme synthesized in E. coli infected with the T4 amber mutant araB22 retains only the 3'- to 5'-nuclease activity (Nossal and Hershfield, 1971). Although the specificity for the appropriate dNTP is thought to be dictated primarily by base pairing to the template strand of the DNA substrate, the enzyme may also be involved. Different mutants of phage T4 in the structural gene for the T4-induced DNA polymerase, gene 43 (see Table 1), exhibit altered mutation rates when compared with the wild-type phage (Speyer et al., 1966, 1968; Drake and Allen, 1968; Drake et al., 1969; Allen et al., 1970; Drake and Greening, 1970). Furthermore, the isolated enzyme from one such temperature-sensitive mutant shows a greater percentage error in the matching of base pairs at the restrictive temperature than does the wild-type enzyme (Hall and Lehman, 1969). However, the observed effect does not quantitatively account for the observed change in mutation rates. Calf thymus DNA polymerase requires the four dNTPs, Mg + +, and a denatured DNA substrate (Bollum, 1959, 1960). Since a DNA primer with a Y-OH-end group is required, the denatured DNA substrate probably loops back on itself (Bollum, 1967). Calf thymus DNA polymerase executes only the repair reaction, yielding a product which closely mimics the substrate DNA (Bollum, 1963a, b). No exonuclease activity is associated with the purified enzyme (Bollum, 1963a, b). A persistent contaminating activity, terminal nucleotidyl transferase, can be separated from the polymerase by gel filtration (BoUum et aL 1964). This latter activity catalyzes the incorporation of dNMP units from dNTPs into the 3'-OH-end of a DNA substrate without using a DNA template (Keir, 1965). The calf thymus DNA polymerase, as well as similar enzymes from other mammalian tissue (Chang, 1971), has a molecular weight of about 100,000 (6-8S), whereas the molecular weight of the terminal deoxynucleotidyl transferase is only about 32,000 (Chang and Bollum, 1971a). Recently, a DNA polymerase has been isolated from rabbit bone marrow and other tissue which has a molecular weight of about 35,000 daltons (Chang and Bollum, 1971b). The lower molecular weight species may come from the nucleus, and the larger from the cytoplasm (Chang and Bollum, 1971b). Several other eukaryotic cell DNA polymerases have been described. A DNA polymerase from Tetrahymena pyriformis has been purified 85-fold (Crerar and Pearlman, 1971); increased activities are found in UV-irradiated ceils (Westergaard and Pearlman, 1969). Two DNA polymerase activities from bakers' yeast can be separated on DEAE-cellulose (Wintersberger and Wintersberger, 1970a). Both prefer a nicked double-stranded DNA substrate, and one has an associated exonuclease activity (Helfman, 1972a). The exonuclease

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DOUGLASW. SMITH

prefers a single-stranded DNA substrate (Helfman, 1972b). Mitochondrial DNA polymerases, of low specific activity, have been purified from rat liver (Meyer and Simpson, 1968, 1970; Kalf and Ch'ih, 1968) and yeast (Iwashima and Rabinowitz, 1969; Wintersberger and Wintersberger, 1970b). The mitochondrial activities differ from those isolated from nuclear fractions, can use denatured and native DNA as substrates, and possess no observable nuclease activity. Net synthesis by the rat liver activity using rat liver mitochondrial DNA was observed (Kalf and Ch'ih, 1968). Spinach chloroplasts contain a similar DNA polymerase (Spencer and Whitfield, 1969). A DNA polymerase from rat liver which requires activated native DNA or poly d(A-T) as DNA substrate has been purified 1000-fold to relative homogeneity (Berger et al., 1971). No terminal deoxynucleotidyl transferase, exonuclease, or endonuclease activity can be detected. Haines et aL (1971) have found distinct cytoplasmic and nuclear DNA polymerase activities in rat liver. The nuclear enzyme, purified 300-fold, has a molecular weight of 65,000, whereas the cytoplasmic enzyme, purified only 30-fold, has an apparent molecular weight of 140,000. A DNA polymerase from sea urchin embryo nuclei of molecular weight about 150,000 has been purified 300-fold to relative homogeneity (Loeb, 1969). It has no observable nuclease activity, but nevertheless prefers native DNA to denatured DNA as a substrate. DNA nicked with mild DNase treatment is an excellent substrate, suggesting the presence of a nick-translation ability (Fig. 14D). The enzyme is found primarily in the nuclei of sea urchin embryos at the hatching stage (about 300 cells per embryo; Loeb et aL, 1969), a period of rapid DNA synthesis. As the embryo approaches the hatching stage, DNA polymerase appears to migrate from the cytoplasm to the nucleus (Fansler and Loeb, 1969; Loeb and Fansler, 1970). A low molecular weight DNA polymerase (21,000 daltons) has been purified 400fold from cultured human KB cells (Greene and Korn, 1970). This enzyme retains an exonuclease activity, and will utilize both native and denatured DNA, as well as nicked DNA, as substrate. During transformation of human peripheral blood lymphocytes, a 30to 150-fold increase in DNA polymerase activity paralleled an increase in DNA synthesis as measured by thymidine incorporation (Loeb et al., 1970; Loeb and Agarwal, 1971). Inhibitor studies suggest that DNA synthesis depends on the increase in DNA polymerase activity. Two enzymes of low specific activity from developing rat brain, differing in their DNA substrate specificity, have been purified about 40-fold (Chiu and Sung, 1971). Two DNA polymerase activities (I and II) have been isolated from HeLa cell nuclei (Schlabach et al., 1971). Activity I can utilize poly d(A-T) and poly (dG:dC) two to four times better than can activity II. Both activities prefer double-stranded DNA containing 3'-OH nicks. A DNA polymerase from Walker 256 carcinosarcoma tissue is inhibited by araCTP when using a DNA or poly (dC :dG) substrate but not when using a poly d(A-T) substrate (Furlong and Gresham, 1971). An enzyme found in both the cytoplasm and nuclear fractions of mouse Ehrlich ascites tumor cells has also been purified 300-fold (Roychoudhury and Block, 1969). It prefers DNase-treated DNA as a substrate, and appears to possess an exonuclease activity. Two polymerase activities have been detected in rabbit kidney ceils infected with Shope fibroma virus, one of which appears to be viral-induced (Chang and Hodes, 1968a, b). The activities can be distinguished immunologically. An activity associated with hepatomas has been found in liver cells. It has a preference for a denatured DNA substrate, whereas the normal liver DNA polymerase prefers a native DNA substrate (Iwamura et al., 1968; Ove et al., 1969a, b). (e) RNA-dependent D N A polymerases. The existence of a DNA stage during the replication of the RNA tumor virus genome was first proposed by Temin (1961). This would

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account for the apparent integration of RNA tumor virus genetic information into the host cell chromosome (Green, 1970; Temin, 1964, 1970; Mizutani and Temin, 1970; Temin and Mizutani, 1970). Enzymes involved in this DNA synthesis might be found in the intact virion, as had already been discovered for RNA polymerase (Kates and McAuslan, 1967). Recently, RNA-dependent DNA polymerase activities were independently discovered associated with Rauscher mouse leukemia virus particles (Temin and Mizutani, 1970) and with Rous sarcoma virus particles (Baltimore, 1970), and the observed DNA synthesis from the viral RNA appears to be necessary for viral transformation (Boettiger and Temin, 1970). Similar activities have subsequently been found in leukemia, sarcoma, and mammary tumor viruses from fowl, murine, hamster, feline, viper and monkey sources (Mizutani and Temin, 1970; Levinson et al., 1970; Rokutanda et al., 1970; Hatanaka et aL, 1970; Green et aL, 1970; Spiegelman et al., 1970b; Salzman, 1971 ; Schlom and Spiegelman, 1971). The activity requires a divalent cation (Scolnick et al., 1970), and the four dNTPs, although up to 40~ activity is observed with only three dNTPs (Temin and Mizutani, 1970). Although the virions contain some DNA (Levinson et al., 1970; Rokutanda et al., 1970; Riman and Beaudreau, 1970), RNase sensitivity suggests that only the RNA is used as a template, and the product DNA specifically hybridizes with the viral RNA (Rokutanda et al., 1970; Spiegelman et al., 1970a; Hatanaka et al., 1971 ; Fujinaga et al., 1970; Garapin et al., 1971). R N A - D N A hybrid molecules are initially formed, followed by formation of duplex DNA molecules (Fujinaga et al., 1970). The product DNA, smaller than the viral RNA (Hatanaka et al., 1970; Spiegelman et al., 1970b), has a similar sedimentation coefficient to the DNA found in the virus (Levinson et al., 1970). Using disrupted virions from which the RNA and DNA has been digested with nuclease, a wide variety of DNA, RNA, hybrid, and synthetic substrates have been tested (Spiegelman et al., 1970b; Spiegelman et al., 1970c; Scolnick et al., 1970; Riman and Beaudreau, 1970). Double-stranded ribopolymers are more active than double-stranded deoxyribopolymers, and there is no incorporation of ribonucleoside triphosphates. Single-stranded polyribonucleotides act as competitive inhibitors (Tuominen and Kenney, 1971). A wide variety of antibiotics have been examined and classified as potential inhibitors of reverse transcriptase activity (Bosmann and Kessel, 1971). In particular, rifampicin and its derivatives inhibit reverse transcriptase (Gurgo et al., 1971; Lancini et al., 1971 ; Crippa and Tocchini-Valentini, 1971 ; Kotler and Becker, 1971). An exciting development was the discovery that lymphoblast extracts obtained from patients with lymphocytic leukemia (Gallo et al., 1970) and extracts from cultured myelocytic leukemia cells (Ackerman et al., 1971) both contained an RNA-dependent DNA polymerase activity. This activity was absent in lymphocyte extracts from nonleukemic patients. These results suggested that leukemic cells might harbor an RNA viral genome. Epithelial cells of patients with the skin disease Xeroderma pigmentosum (Cleaver, 1970) also appear to contain an RNA-dependent DNA polymerase activity (Miiller et al., 1971). Such cells are deficient in a DNA excision-repair system (Cleaver, 1968; Jung and Schnyder, 1970; Setlow et aL, 1969). The reverse transcriptase activity is absent in epithelial cells from normal patients, suggesting that the disease may be due to an oncogenic RNA virus. C-type viruses have been described in avian, mammalian, and reptilian species (Dmochowski and Grey, 1957; Huebner and Todaro, 1969). Reverse transcriptase activity has been found in several of these viruses (Aaronson et aL, 1971 ; Parks et al., 1972; Theilen et al., 1971 ; Kawakami et al., 1971), including those derived from two human tumors, RD114 (McAllister et al., 1971) and ESP-1 (Priori et aL, 1971; Gallo et aL, 1971). The type C virus from human origin is antigenically distinct from other mammalian type C viruses

370

DOUGLAS W. SMITH

(Gallo et al., 1971), and considerable circumstantial evidence suggests that these viruses may play a role in human cancer (Dmochowski, 1969). Further, the reverse transcriptase from primate C-type viruses is immunologically distinguishable from those of lower animal Ctype viruses (Scolnick et al., 1972). However, RNA-dependent DNA polymerase activity does not appear to be an intrinsic property of RNA tumor viruses. Scolnick et al. (1971), and others (Weber et al., 1971; Bosmann, 1971 ; Penner et al., 1971 ; Coffin and Temin, 1971), discovered that uninfected nonmalignant cells contain reverse transcriptase activity. Such activity has also now been found associated with nononcogenic viruses such as visna virus (Schlom et al., 1971; Scolnick et al., 1970; Stone et al., 1971; Parks et al., 1971; Lin and Thormar, 1970). A recent autoradiographic study has implicated an RNA-dependent DNA polymerase in the amplification of ribosomal DNA in Xenopus oocytes (Ficq and Brachet, 1971). Reverse transcriptase actiyity is increased during gene activation effected by phytohaemagglutinin in human lymphocytes (Penner et al., 1971). Rat liver mitochondria appear to contain a reverse transcriptase activity (Bosmann, 1971). Thus, the presence of an RNA-dependent DNA polymerase activity does not imply the necessary presence of an RNA viral genome, and reverse transcriptase may be involved in many biological events. It is clear that the described activities are probably due to a heterologous group of enzymes. The avian and mammalian enzymes are in fact differentially inhibited by ethidium bromide (Fridlander and Weissbach, 1971), and the reverse transcriptase activities of the C-type viruses can be immunologically distinguished (Scolnick et al., 1972). A common experimental practice has been to utilize synthetic nucleic acid substrates such as poly(rA).poly(rU) or poly(rA).poly(dT). It is not known that the reverse transcriptase enzymes utilize these artificial substrates in the same way as they would the in vivo nucleic acid substrate, and there is evidence that the viral-induced enzymes prefer a double-stranded RNA substrate, such as poly(rA).poly(rU), whereas the endogenous enzyme prefers a hybrid nucleic acid substrate, such as poly (rA).poly(dT) (Spiegelman et al., 1970c; Ross et al., 1971; Scolnick et al., 1971). The optimal substrate for both appears to be a hybrid molecule with a long polyribonucleotide as template and small oligodeoxynucleotides as primers (see Fig. 13B), such as poly(rA).oligo(dT) (Baltimore and Smoler, 1971; Hatanaka et al., 1971). A repair synthesis is observed, with no extensive synthesis. Thus, the reverse transcriptase appears to execute a repair-type synthesis, using a polyribonucleotide as template and a short polydeoxyribonucleotide as primer. One obvious possibility for the source of the primer DNA is the viral DNA (see above), although this has not yet been shown. An enzyme from E. coli which preferred an R N A - D N A hybrid substrate was described some time ago (Lee-Huang and Cavalieri, 1963). Further, E. coli DNA polymerase I can use RNA (Cavalieri and Carroll, 1970a, 1971) or poly (rA).poly(dT) (Karkas et al., 1972) as a substrate. The RNA virus reverse transcriptase can be distinguished from E. coli DNA polymerase I and from the calf thymus DNA polymerase on the basis of substrate specificity (Goodman and Spiegelman, 1971). The reverse transcriptase from avian myeloblastosis virus (AMV) has been purified 40fold, to 90~ homogeneity (Kacian et al., 1971). It possesses two subunits, of molecular weights 110,000 and 69,000, exhibits no RNA or DNA endonuclease activity, and can utilize double-stranded RNA, double-stranded DNA, or RNA-DNA hybrid nuclei acids as substrates. However, Moiling et al. (1971b) find co-purification of a nuclease called RNase H with the DNA polymerase from AMV (Moiling et al., 1971a). RNase H specific-

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ally degrades the RNA strand of an RNA-DNA hybrid molecule (Stein and Hausen, 1969; Hausen and Stein, 1970). Maia et aL (1971)have purified the reverse transcriptase from chick embryos 1000-fold, and find a single subunit of molecular weight 50,000 daltons. The enzyme is distinct from two DNA-dependent DNA polymerases, and is nearly specific for an R N A - D N A hybrid substrate (Maia et al., 1971; Stavrianopoulos et al., 1971). The DNA polymerase of Rous sarcoma virus utilizes the 60S viral RNA better than any other RNA as a substrate, but can use heat-dissociated viral RNA annealed to oligo(dT) or oligo(dC) 20 to 30 times better (Duesberg et aL, 1971a, b; Bishop et aL, 1971). The enzyme may prefer a partially double-stranded, or hybrid region, of an RNA species for optimal DNA synthesis. E

(Mornrnalion '~ \phogeinduced,/

E

(E.coli)

+ ATP ~

E - A M P +PPi

(I) + NAD~

E-AMP+NMN

-q -P---F (2)

E-AMP

Jr

~-OH_O~0..~ "lOi o-

E o

=,

"

.~-OH ~/~1oO~1.~ ~,o -°~ ~'RA

P---F-

+

E

+ AMP

o

I

o--P~o RA

FIo. 16. Postulated mechanism of the reactions catalyzed by the D N A ligases. See the text for details. Figure from Olivera et al. (1968b) and Richardson (1969).

2. D N A ligases

These enzymes and their reactions have been reviewed (Richardson, 1969; Goulian, 1971). A phosphodiester bond is formed between a 5'-phosphoryl group and a 3'-hydroxyl group at the ends of DNA strands (Gefter et aL, 1967; Olivera and Lehman, 1967a; Zimmerman et al., 1967; Weiss et al., 1968a). The ends to be joined must be in juxtaposition, usually effected by mutual base pairing with a common complementary DNA strand (see Fig. 16). Although T4 ligase can join DNA duplexes at their base-paired ends (Sgaramella et al., 1970), double-stranded polyribonucleotides are not joined. However, nicks in either strand of a polydeoxyribonucleotide-polyribonucleotide substrate can be joined by ligase (Kleppe et al., 1970). DNA ligase from E. coli (Gefter et al., 1967; Gellert, 1967; Olivera and Lehman, 1967a) requires NAD as a cofactor (Zimmerman et aL, 1967; Olivera and Lehman, 1967b). However, the enzymes from E. coli infected with bacteriophages T2 (Weiss and Richardson, 1967), T4 (Cozzarelli et al., 1967), or T7 (Becker et al., 1967), from mammalian cells (Lindahl and Edelman, 1968; Spadari et al., 1971) and virus-infected mammalian cells (Sambrook and Shatkin, 1969), or from higher plants (Kessler, 1971 ; Howell and Stem, 1971), require ATP as cofactor (Weiss and Richardson, 1967; Becker et al., 1967; Lindahl and Edelman, 1968). The postulated reaction mechanism involves three steps (Fig. 16). The cofactor is co-

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DOUGLAS W. SMITH

valently joined to the enzyme in the first step. Evidence for this includes the following: E. coli DNA ligase will catalyse an exchange between N M N and NAD, but not between AMP and NAD (Olivera et ak, 1968b; Little et aL, 1967), and the T4-induced enzyme will catalyse a similar exchange between pyrophosphate and ATP, but not between AMP and ATP (Becker et aL, 1967; Lindahl and Edelman, 1968; Weiss and Richardson, 1967; Weiss et aL, 1968a). In addition, acid and alkali stable ligase-AMP complexes have been purified from E. coli (Olivera et aL, 1968b; Little et aL, 1967) and from T4-infected E. coli (Becker et al., 1967; Weiss and Richardson, 1967; Weiss et aL, 1968a). The AMP is covalently joined to the amino acid of a lysine residue in a phosphoamide linkage, in both the E. coli ligase and the T4-induced ligase (Gumport and Lehman, 1971). Upon incubation with the appropriate DNA substrate, AMP is released from this complex (OIivera et al., 1968b), and the complex will catalyse phosphodiester bond formation in the absence of added cofactor (Little et al., 1967; Weiss et al., 1968a). In the second step in the reaction mechanism, shown for the E. coli (Olivera et aL, 1968a) and T4-induced (Harvey et al., 1971) enzymes the AMP moiety is transferred from the ligase-AMP complex to the 5'-phosphoryl group of the DNA substrate (Fig. 16), with formation of a pyrophosphate linkage. The final step is a ligase catalysed formation of the phosphodiester bond with release of AMP. Both the E. coli DNA ligase and the T4-induced enzyme (Hall and Lehman, 1969) will catalyse this latter reaction. Early assay procedures include conversion of hydrogen-bonded phage lambda DNA circles (Hershey et aL, 1963) into covalently joined circles (Gellert, 1967). This procedure has been modified to assay the covalent joining of a radioactive lambda DNA molecule hydrogen-bonded to a second, cross-linked lambda DNA molecule. Joining is assayed by observing conversion of radioactive DNA into a rapid-renaturable form following alkaline denaturation (Zimmerman et aL, 1967). In another somewhat similar assay, the nonradioactive DNA molecule is covalently bound to a cellulose matrix, permitting isolation via sedimentation or filtration. Ligase is assayed by measuring covalent joining of a second radioactive DNA molecule to this immobilized DNA molecule (Cozzarelli et aL, 1967; Bertazzoni et al., 1971). A second procedure assays conversion of phosphatase-sensitive p32-1abeIed DNA phosphate groups into phosphatase-resistance groups, using either DNA (Gefter et aL, 1967; Weiss et al., 1968b) or p32-1abeled oligo(dT) hydrogen-bonded to a poly(dA) chain (Olivera and Lehman, 1967a). The 5'-phosphoryl groups in single-strand nicks in a DNA substrate are labeled with p32 via a phosphatase-polynucleotide kinase procedure (Weiss et aL, 1968c). More recent assays include the intra-strand joining of the ends of small oligo d(A-T) polymers, forming single-stranded circles (Modrich and Lehman, 1970), and formation of the ligase-AMP intermediate via conversion of radioactive NAD into an acid-insoluble form (Zimmerman and Oshinsky, 1969). Assay of formation of the ligase-AMP intermediate has led to identification of three forms of the E. coli DNA ligase (Zimmerman and Oshinsky, 1969). In low salt, the presumed native form III, of molecular weight about 100,000 daltons, is converted to a form II, which is unable to catalyse step 3 and has reduced activity in step 2 (Fig. 16). Form II then is converted to a form I, of molecular weight about one-half that of form III, which is capable of catalysing only the exchange reaction between N M N and NAD. 3. Deoxyribonucleases ( DNases)

Properties of many nucleases have been extensively reviewed (Lehman, 1963, 1967;

D N A SYNTHESIS IN PROKARYOTES: REPLICATION

373

Richardson, 1969; Radding, 1969; Arber and Linn, 1969; Koerner, 1970; Echols, 1971; Goulian, 1971 ; Signer, 1971). Only the basic properties of some of these will be discussed below. (a) Bacterial DNases. Several exonucleases and endonucleases can be isolated from E. coli extracts. The first isolated nuclease, exonuclease I, is specific for single-stranded DNA, degrading from the 3'-end and releasing 5'-deoxymononucleotides (Lehman, 1960; Lehman and Nussbaum, 1964). Exonudease II is now the name given to both nuclease activities associated with the polypeptide chain called DNA polymerase I; its properties were discussed above. Exonuclease lII, a relatively small protein, is specific for double-stranded DNA, degrading both strands from the 3'-end and releasing 5'-deoxymononucleotides (Richardson et al., 1964c). Unlike exonuclease I, exonuclease III can act initially as a phosphatase, releasing inorganic phosphate from a DNA substrate with a 3'-phosphoryl group, before performing its exonucleolytic function (Richardson and Kornberg, 1964; Richardson et al., 1963). As discussed above, an exonuclease III-treated DNA molecule is an excellent substrate for the E. coli DNA polymerase enzymes. An exonuclease IV, showing a preference for oligonucleotides over either single-stranded or double-stranded DNA as a substrate, has also been described (Jorgensen and Koerner, 1966). Endonuclease I, a small enzyme found in the periplasmic region of the cell wall (Cordonnier and Bernardi, 1965), constitutes about 90~o of the total E. eoli nuclease activity (Shortman and Lehman, 1964). It hydrolyses either single-stranded or double-stranded DNA to oligonucleotides (Lehman et al., 1962a) in a reaction competitively inhibited by tRNA (Lehman et al., 1962b). With double-stranded DNA, double-strand breaks, or "chops", are introduced into the DNA molecule (Bernardi and Cordonnier, 1965; Studier, 1965), as opposed to a single-strand break or "nick". Endonuclease II (Friedberg and Goldthwait, 1968, 1969) requires double-stranded DNA, can degrade partially depurinated T4 DNA (Hadi and Goldthwait, 1971), and prefers alkylated DNA, introducing a limited number of single-strand nicks into the substrate. The role of these enzymes in DNA replication remains unknown. Mutants of endonuclease I and exonuclease II (DNA polymerase I) are discussed above; these mutants are viable, indicating that these enzymes are dispensable. Two other endonucleases, involved in the restriction of phage DNA by certain E. coli strains in vivo, have been purified and discussed (Arber and Linn, 1969). An ATP-dependent exonuclease, initially detected by Buttin and Wright (1968) and Willetts et al. (1969), has been purified from E. coli ree + cells; this enzyme appears to be absent in E. eoli recB or reeC mutants (Oishi, 1969; Wright and Buttin, 1969; Barbour and Clark, 1970; Wright et al., 1971). 0.2 mM ATP and pH 9 are optimal, and dATP can substitute for ATP. R e e B and reeC mutants complement each other in vivo, but not in vitro; there is no further decrease in activity in a reeBrecC double mutant (Barbour and Clark, 1970). Wright et aL (1971) have shown the presence of two exonuclease activities. The first, exonuclease V, requires noncircular double-stranded DNA and yields 3'-OH- and 5'-phosphoryl oligodeoxynucleotides, which become smaller in time. The second degrades single-stranded DNA from the 3'-end in the absence of ATP; heat inactivation distinguishes this activity from exonuclease I. Goldmark and Linn 0970), assaying exonuclease [ sensitivity of single-stranded circular phage fd DNA, have purified an ATP-dependent endonuclease activity 10,000-fold from E. coil This activity also is absent in extracts of reeB or recC mutants. 0.3 mM ATP and pH 7 are optimal; dATP can only partially substitute for the ATP. However, reconstruction experiments using combined E. eoli reeB and tee + extracts indicate the presence of a heat-sensitive inhibitor in the recB or recC extract; this

374

DOUGLAS W. SMITH

inhibitor appears to be absent in the ree + extracts. Recent evidence (Goldmark, personal communication) suggests that the recB, C nuclease possesses four enzymatic activities, including the above mentioned two exonuclease and one endonuclease activities, plus an ATPase activity. The latter activity has been purified 3500-fold (Nobrega et aL, 1972). It requires DNA and prefers native DNA, copurifies with the exonuclease activity, and is inactivated by heat at the same rate as the exonuclease. Stoichiometrically, 8 to 9 ATP molecules are hydrolyzed to ADP and phosphate for each DNA phosphodiester bond broken. Recently, Ebisuzaki et aL (1972) have purified two DNA-dependent ATPase activities from E. coli and one from T4-infected E. coli by assaying ATP hydrolysis. No DNA exonuclease or endonuclease activity is observed with these activities. A similar ATPase activity associated with an ATP-dependent DNase from Micrococcus luteus has been described (Anai et aL, 1970a, b, 1971). An ATP-dependent DNase which yields mono- and oligodeoxynucleotides and ADP has been partially purified from Mycobacterium smegmatis (Winder and Lavin, 1971). Loss of an ATP-dependent DNase appears to occur upon infection of E. coli with phage T4; T4-induced protein synthesis is required for this loss (Yamazaki, 1971). (b) Phage-induced DNases. Several nucleases are induced following infection with the Teven phages; these and other phage-induced enzymes have been reviewed (Kozloff, 1968; Aposhian, 1968; Koerner, 1970; Cohen, 1968; Goulian, 1971). Endonuclease II (Sadowski et al., 1968; Sadowski and Hurwitz, 1969a) introduces about one single-strand nick per 1000 nucleotides into double-stranded DNA, and endonuclease IV (Sadowski et aL, 1968 ; Sadowski and Hurwitz, 1969b; Ling, 1971) attacks single-stranded DNA, yielding oligonucleotides approximately 150 nucleotides in length. Since these are both early enzymes and do not attack T4 DNA, they are thought to be involved in host DNA degradation. Endonuclease II would first nick the host DNA; these nicks would be extended into "gaps" of single-stranded DNA by an as yet unidentified exonuclease; endonuclease IV would then attack these"gaps", leaving small double-stranded oligonucleotides. Such products are found following infection with T4 mutants in genes 46 and 47; the gene products of the latter genes may be exonucleases involved in further host DNA degradation (Short and Koerner, 1969; Koerner, 1970). Mutants of endonuclease II have been isolated (Warner et aL, 1970; Hercules et al., 1971), and this enzyme may be identical with a similar activity, endonuclease B, described by Ando et al. (1969). The latter authors described a second activity, endonuclease A, specific for double-stranded DNA, yielding nicks having only deoxyguanylate residues on the 3'-end of the nicked DNA. Late in infection, yet another endonuclease activity, specific for double-stranded DNA and active on T4 DNA, appears (Altman and Meselson, 1970). Two exonuclease activities have been purified from T4 infected cells. One is associated with the T4-induced DNA polymerase discussed above. The other, exonuclease A, hydrolyses nicked double-stranded DNA from the 3'-OH-ends, liberating 5'-deoxynucleotides (Short and Koerner, 1969). Exonuclease A is present in cells infected with gene 46 or 47 T4 mutants. A DNA-dependent ATPase is found in T4-infected ceils (Debrecini et al., 1970); ATP is hydrolysed to ADP and inorganic phosphate in the presence of all DNA species tried except native T4 or T7 DNA. No known T4 mutants lack this activity. Genes 3 (Center et al., 1970; Center and Richardson, 1970a, b; Sadowski, 1971) and 6 (Kerr and Sadowski, 1972a, b) of bacteriophage T7 code for deoxyribonucleases. The gene 3 product, endonuclease I, has been purified 1800-fold. It degrades both single-stranded and double-stranded DNA, introducing both single-strand "nicks" and double-strand "chops" into double-stranded DNA. Extensive digestion of T7 DNA results in about 600 breaks per

D N A SYNTHESIS IN PROKARYOTES; REPLICATION

375

strand (Center and Richardson, 1970b). The gene 6 product is an exonuclease, which has been purified 500-fold (Kerr and Sadowski, 1972a). Thermosensitive gene 6 T4 mutants yield a thermosensitive enzyme. The enzyme requires Mg + ÷, is stimulated by K ÷, and prefers a double-stranded DNA substrate. It hydrolyses duplex DNA until about 50~ of the DNA is acid soluble (Kerr and Sadowski, 1972b), leaving single-stranded DNA and 5'mononucleotides as products of the reaction. It begins hydrolysis at the 5'-termini of the DNA, releasing only mononucleotides if the DNA contains a 5'-phosphoryl terminus and releasing an initial dinucleoside monophosphate if the DNA contains a 5'-hydroxyl terminus. Hydrolysis can be initiated at nicks, as well as at external termini. Both of these nucleases are involved in degradation of host cell DNA, and may also be involved in genetic recombination. A second T7-induced endonuclease, endonuclease II, has been purified 400-fold (Center, 1972). This enzyme has no activity toward single-stranded DNA, and introduces nicks with 3'-OH and 5'-phosphoryl ends into double-stranded DNA. A maximum of thirteen breaks per molecule are introduced into phage T7 DNA. A T5-induced DNase has been purified 400-fold, and shown to degrade single-stranded or double-stranded DNA from the 5'-end, releasing 5'-phosphoryl oligodeoxynucleotides of average length three residues (Frenkel and Richardson, 1971a). No endonuclease activity is observed, and UV-irradiated DNA will serve as a substrate. A thermosensitive phage T5 mutant in the gene for this enzyme has been obtained (Frenkel and Richardson, 1971b). No plaques form at 45 °, and DNA synthesis is prematurely arrested. Conversion of rapidly sedimenting DNA forms into mature length phage DNA appears to be defective. The gene products of phage lambda genes O and P remain unknown; however, indirect evidence suggests that one (or both) may be an endonuclease capable of nicking twisted circular DNA (Shuster and Weissbach, 1968a, b, 1969; Freifelder and Kirschner, 1971). A lambda exonuclease (Korn and Weissbach, 1963) has been purified and characterized (Radding, 1966; Little, 1967). It preferentially utilizes double-stranded DNA, degrades progressively from the 5'-end, requires a 5'-phosphoryl group, and releases 5'-dNMPs. The enzyme binds to nicked circular double-stranded DNA, but does not release nucleotides; thus, it cannot degrade at a nick (Carter and Radding, 1971; Masamune et aL, 1971a). The enzyme is a multimer of identical subunits of molecular weight 24,000 daltons (Radding and Carter, 1971), and is involved in the lambda general recombination system (Signer, 1971; Cassuto and Radding, 1971). 4. Denaturation-renaturation proteins Using a procedure based on binding to DNA-cellulose, the gene product of phage T4 gene 32 has been purified (Alberts et al., 1968; Alberts, 1970). Gene 32 is required for DNA replication (Epstein et al., 1963) and for recombination (Tomizawa et al., 1966), suggesting an essential role for 32-protein in these processes. The product is a single polypeptide chain with a molecular weight of about 35,000 daltons and the shape of an elongated prolate ellipsoid (Alberts and Frey, 1970). About 10,000 molecules are found in T4-infected cells, both at early and at late times of infection. These are required in stoichiometric amounts rather than in catalytic amounts by T4-infected cells (Snustad, 1968; Sinha and Snustad, 1971). The protein binds very strongly to single-stranded DNA (dissociation constant of 10 -9 M) in a highly cooperative reaction. Clustered binding occurs under non-saturating conditions, with a binding of one 32-protein molecule per 10 nucleotides at saturation. Thus, a highly extended DNA-protein complex of unusually low sedimentation coefficient is formed (Alberts and Frey, 1970). The strong binding to single-stranded DNA causes

376

DOUGLAS W. SMITH

32-protein to promote denaturation of double-stranded DNA, thus lowering the melting temperature (Tin) of the native DNA. For example, the Tm of poly d(A-T) is lowered from about 65 ° to about 25° by the presence of 32-protein (Alberts and Frey, 1970). On the other hand, 32-protein also promotes renaturation, presumably by prevention of intrastrand hydrogen-bonding in the single-stranded DNA species. The role of 32-protein in DNA replication remains unknown, although it may act to promote unwinding of the parental DNA helix ahead of the replication fork (Alberts and Frey, 1970) and specific models have been proposed (Alberts, 1971). In in vitro experiments, 32-protein promoted the rate of synthesis by T4 DNA polymerase on a single-stranded DNA substrate, but the synthesis using E. coil DNA polymerase I was unaltered. A similar protein has been purified from E. coil (Alberts, pers. comm.) and appears to stimulate DNA polymerase II activity in vitro (Gefter, pers. comm.). This protein has a molecular weight of about 25,000 daltons, is present at about 700 molecules per bacterium, and at saturation about one protein molecule is bound per 10 nucleotides of the DNA substrate. Another protein similar to the T4 gene 32-protein has been isolated using DNA-cellulose columns from E. coli infected with the filamentous bacteriophages fd or M13 (Alberts et aL, 1972). The protein is altered in phage gene 5 mutants, and hence is the gene 5 product. The protein has a molecular weight of about 10,000 daltons, is present in about 100,000 copies per cell, and binds tightly and cooperatively to single-stranded DNA but not to doublestranded DNA. At saturation, about one protein is bound per 4 DNA nucleotides. This selective binding again promotes denaturation of helical DNA. However, unlike the 32protein, which forces the DNA into an extended linear configuration, the M13 gene 5protein causes two protein-covered DNA strands to come together, forming a helical, rodlike structure. Thus, the two proteins may have different physiological roles in phageinfected cells. 5. Omega protein

An activity able to remove superhelical twists from lambda DNA closed circles has been isolated from E. coli (Wang, 1971). Protease- and heat-sensitivity suggest that the molecule, called omega, is a protein. Omega protein has no detectable DNA ligase activity, and has a molecular weight of about 100,000 daltons (Wang, pers. comm.). Omega binds preferentially to single-stranded DNA, and removes supertwists only from negatively twisted closed circular DNA. The formation of single-stranded regions in such DNA is favored thermodynamically (Bauer and Vinograd, 1970), and may explain the preference of omega for such a substrate. The mechanism for the omega catalysed reaction is not understood, but Wang (1971) favors a sequential nicking of the twisted DNA by omega, with a subsequent covalent linkage formed between omega and the 5'-phosphoryl group in the nick. This would be followed by unwinding of one or more supertwists, with subsequent closure of the nicked DNA and release of omega. The physiological role of omega is unknown, but the above mechanism suggests that omega may be a swivel protein for unwinding DNA during replication. A similar activity, able to unwind either positively or negatively twisted closed circular DNA, has been detected in secondary mouse embryo cells (Champoux and Dulbecco, 1972). A somewhat similar enzyme may also have been detected in a proteinDNA complex (Green et al., 1971b) isolated from polyoma virus-infected secondary mouse cells (Bourgaux and Bourgaux-Ramoisy, 1972).

D N A SYNTHESIS IN PROKARYOTES: REPLICATION

377

6. Plasmid DNA-protein complex Colicinogenic factor El (Col El) can be isolated as a DNA-protein complex from Brij 58 lysates of E. coli (Clewell and Helinski, 1969, 1970c). The Col E1 DNA has properties of the superhelical form I. Upon treatment with heat, detergents, alkali, phenol, or proteases, the complex "relaxes", i.e., some protein is freed and the DNA is converted into the nicked circular form II. Similar complexes containing colicinogenic factors Col Ez (Clewell and Helinski, 1970a; Blair et al., 1971), Col Ea (Clewell and Helinski, 1970a), the colicinogenic factor-sex factor, Col Ib (Clewell and Helinski, 1970b), and the sex factor F1 (Kline and Helinski, 1971), have been isolated. However, for these plasmids heat (60 ° for I0 min) does not relax the complex, but, on the contrary, protects the complex from relaxation by other treatments, e.g., by detergent or proteases (Clewell and Helinski, 1970a; Helinski, 1970). This suggests the presence of a nuclease which normally nicks the form I DNA but is heatsensitive in all but the Col E~ complex (Helinski, 1970); however, the putative nickase has not been isolated in an active form. Alternatively, the protein may be immobilizing the two ends of an already nicked form II DNA, causing it to behave as form I in the complex; relaxation would occur simply by release of the two arms. However, this possibility does not easily explain the results of heat treatment of the Col E~, Col Ez, and F~ complexes. Using poly (U,G) binding and CsCI gradient analysis of the separated col factor DNA strands (Vapnek and Rupp, 1970; see Fig. 3), it has been shown that the DNA strand which is nicked is unique. This is true for the Col E~ and Col E2 complexes (Blair et al., 1971) and for the sex factor F1 complex (Kline and Helinski, 1971).

D. In vitro DNA Synthesis Systems l. Permeable cell systems As studies with the isolated E. coli DNA polymerase I progressed during the 1960s, it became increasingly clear that this enzyme alone could not replicate DNA. Attempts to isolate other polymerizing enzymes by classical enzymological procedures were unsuccessful. Evidence was also slowly accumulating that DNA replication is a complex process, probably requiring several enzymes and a structural "site" within the cell. A replication "complex" was visualized in which the replicating DNA, small molecule substrates and cofactors, enzymes and other protein factors would be spatially oriented and regulated in a structural site, permitting DNA replication to proceed with the extraordinary fidelityand speed observed in vivo. It appeared that integrity of such a complex might be the necessary requirement for development of a successful in vitro DNA replication system. The simplest approach is to perturb intact ceils as little as possible, and still permit in vitro experiments, that is, permit incorporation of the immediate precursors of DNA replication. Several methods have now been successfully used to render bacterial cells permeable to phosphorylated derivatives of the deoxyribonucleosides. Treatment of E. coli with the chelating agent, ethylenediaminetetraacetic acid (EDTA), alters the cell permeability properties and releases the enzymes found in the periplasmic space (Neu and Heppel, 1965). Either tris-EDTA or tris-Mn ÷ ÷ treated E. coli cells, when placed in a Mg ÷ +phosphate reaction mixture, will incorporate dNTPs into DNase-sensitive macromolecules (Buttin and Kornberg, 1966; Buttin and Wright, 1968). However, the observed DNA synthesis does not proceed in a semi-conservative manner, and appears to be dependent on the presence of nicks introduced into the DNA by endonuclease activity. No synthesis is P.S. 26---N

378

DOUGLAS W. SMITH

o b s e r v e d in t r i s - E D T A treated E. coli m u t a n t s deficient in endonuclease I (Buttin and Wright, 1968). T r e a t m e n t with any o f several organic solvents, including c h l o r o f o r m , toluene, o r ether, alters the p e r m e a b i l i t y p r o p e r t i e s o f bacteria. E t h e r - t r e a t e d cells (Vosberg a n d H o f f m a n Berling, 1971; D i i r w a l d a n d Hoffmann-Berling, 1971), a n d t o l u e n e - t r e a t e d cells (Moses a n d R i c h a r d s o n , 1970a) have been extensively studied. The basic features o f these p e r m e a b l e cell systems are very similar, a n d are discussed together. In the presence o f K +, M g ÷ +, and

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FIG. 17. Density-labeled DNA synthesis in vitro: ATP dependence. E. coli HF4702 thy-dap-endoI- polA ÷ (,~)was grown in tris-glucose-casamino acids medium containing diaminopimelic acid (dap) and l*C-thymine to 2 × 108 cells/ml, harvested, and resuspended in 4 % agarose. The gelled agar was fragmented, and 3 hr spheroplasts were formed in penassay broth-sucroseMg ÷ + medium lacking dap. The spheroplasts were lysed, and the agar framents were resuspended in 0.3 ml reaction mixture and incubated at 37° for 30 min. This mixture was similar to that shown in Table 3, except that dBUTP was substituted for TTP, 3H-dATP for 3H-CTP, and AdR for CdR. The DNA was extracted using a KI-pronase-sarkosyl procedure (Smith et al., 1970), and centrifuged to equilibrium in a CsCI density gradient to which E. coli DNA was added as an optical density marker. --O--~4C-thymine prelabel; - - © - - a l l in vitro label; - - × - - o p t i c a l density marker. A T P , the f o u r d N T P s are i n c o r p o r a t e d into D N A at a rate s o m e w h a t less than the in vivo rate for at least 60 min. W h e n p r o v i d e d with a p p r o p r i a t e precursors, ether-treated cells can also synthesize R N A a n d p r o t e i n (Vosberg a n d Hoffman-Berling, 1971). The synthesis in the presence o f A T P is semi-conservative whereas that observed in the absence o f A T P is non-conservative, as shown by density-labeling experiments with d B U T P substituted for TTP. A n e x a m p l e o f s u c h an e x p e r i m e n t is shown in Fig. 19A, using toluene-treated B. s u b t i l i s cells ( M a t s u s h i t a et al., 1971). The in vitro synthesized D N A is biologically active in a t r a n s f o r m a t i o n assay system (Fig. 19B). Synthesis requires all four d N T P s a n d M g + +, a l t h o u g h M n ÷ ÷ can p a r t i a l l y substitute

D N A SYNTHESISIN PROKARYOTES: REPLICATION

379

for Mg + +. Na + will not substitute for K +. Other ribo- and deoxyribonucleoside triphosphates will partially substitute for the ATP requirement, r, 7-methylene-ATP will not substitute for ATP (Vosberg and Hoffman-Berling, 1971), indicating that the function of the ATP is more fundamental than simply maintenance of the dNTP concentrations. Ethertreated cells will incorporate dNMPs (Vosberg and Hoffman-Berling, 1971), but toluenetreated cells will not (Moses and Richardson, 1970a). Both systems will incorporate dNDPs. Semi-conservative synthesis is specifically inhibited by the sulfhydryl inhibitors, N-ethylmalimide (NEM) and p-hydroxymercuribenzoate (pHMB). NAD, cyanide, and azide have no effect on these systems. Semi-conservative, or replicative, synthesis is observed in permeabilized p o l A - or endoI- E. coli mutants, is insensitive to antibody against E. coli DNA polymerase I, and is thermosensitive in thermosensitive dna mutants (Moses and Richardson, 1970a; Mordoh et al., 1970; Kohiyama and Kolber, 1971 ; Hirota et al., 1972; Vosberg and Hoffman-Berling, 1971). The ATP-independent non-conservative synthesis is NEM-insensitive, is present at the restrictive temperature using E. coil dna mutants, and is stimulated by endogenous or exogenous nuclease activity. In toluene-treated cells, its demonstration requires a p o l A + strain (Moses and Richardson, 1970a). In ether-treated hcr- E. coli cells previously infected with phage $ × 174, the host DNA synthesis is sensitive to mitomycin C treatment, but the phage DNA synthesis is insensitive, analogous to in vivo observations (Vosberg and Hoffman-Berling, 1971). Phage DNA synthesis yields primarily RFI molecules, with both DNA strands labeled. No ~b× 174 RF DNA is synthesized in vitro with ether-treated E. coli infected with gene A ~b× 174 mutants. Replicating molecules that appear to be longer than unit length are also obtained (Diirwald and Hoffman-Berling, 1971). In pulse experiments, short chains (about 10S) appear before long chains, and under conditions of limiting dNTP concentrations, even shorter pieces (about 5S) are obtained (Geider and Hoffman-Berling, 1971). Density labeling experiments show the presence of some newly synthesized DNA covalently attached to pre-existing parental DNA, in addition to the usual semi-conservative synthesis (Geider and HoffmanBerling, 1971). Repair synthesis is not thought to be the source of this DNA product. The toluene treatment has been adapted to B. subtilis (Matsushita et al., 1971) and used to show that the in vitro synthesis is semi-conservative and yields a biologically active product (Fig. 19). Furthermore, synthesis proceeds at the replication forks existing in the cells prior to toluene treatment. Burger (1971) has also presented evidence using toluene-treated E. coli that the in vitro synthesis proceeds only at pre-existing replication forks. Fleischman and Richardson (1971) have used toluene-treated cells to show that hydroxymethyldeoxycytidine-5'-triphosphate (dHMCTP) is specifically not incorporated into DNA in E. coil restrictive to nonglucosylated T-even phage, but is incorporated into DNA using permissive E. coll. Thus, T-even phage enzymes are not needed for incorporation of dHMCMP into DNA, and the host restriction system can recognize hydroxymethylcytosine (HMC) residues in host DNA, as well as in phage DNA. Billen et al. (1971a, b) observed that freezing B. subtilis cells in liquid nitrogen, with subsequent rapid thaw at 37 °, rendered the cells permeable to deoxytriphosphates. The treated cells could still incorporate thymidine into DNA. Density-labeling experiments indicated that the triphosphates entered DNA via a non-conservative synthesis, whereas the thymidine entered DNA via semi-conservative synthesis. UV-irradiation enhanced the non-conservative synthesis, but reduced the semi-conservative synthesis. It was suggested that the two synthesizing systems exist as separate, nonmixing "compartments" in the cell.

380

DOUGLASW. SMITH

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F r a c t i o n number FIG. 18. Synthesis of infectious phage ¢ x 174 D N A using an in vitro membrane system. The cell-free in vitro membrane system was prepared by gently lysing 32p_~ x 174-infected E. coli H560 (W3110 polAl endoI- thy-) using lysosyme-EGTA-Brij 58 and centrifuging the rapidlysedimenting (see Fig. 9) membrane-DNA complex in a sucrose gradient to the "shelf" of a 6 0 ~ sucrose pad. The fractions from this gradient containing the membrane-DNA complex were incubated at 30 ° for 20 min in a typical reaction mixture (see Table 3) with dBUTP substituted for TTP. The DNA was extracted with pronase and sarkosyl and the ¢ × 174 DNA purified using a 5 ~ - 2 0 ~ sucrose gradient with a CsCI pad. Fractions containing the RFII x 174 DNA were combined, brought to a density of 1.73 g/cc with CsC1 and centrifuged for 55 hr at 34,000 rpm. 43 fractions were collected into 1 ml sterile tris-EDTA buffer, and sampled for infectivity using the Guthrie and Sinsheimer (1963) spheroplast assay. The remainder of the fractions were acid-precipitated, collected on Millipore filters, and assayed for radioactivity using a liquid scintillation counter. Figure from Knippers and Str/itling (1970).

G a n e s a n (1971) has d e s c r i b e d a p e r m e a b l e cell s y s t e m for B. subtilis in w h i c h ceils a r e f r o z e n in l i q u i d n i t r o g e n , t h a w e d , a n d i n c u b a t e d at 0 ° in a s u c r o s e - M g ÷ + m i x t u r e c o n t a i n ing 0.05 M s o d i u m a z i d e a n d Brij 58. A f t e r 2 to 3 hr, 5 5 ~ o f the c e l l u l a r p r o t e i n a n d 95~o o f the D N A p o l y m e r a s e a c t i v i t y is lost, a n d the cells b e c o m e p e r m e a b l e to d e o x y n u c l e o s i d e t r i p h o s p h a t e s a n d to d e o x y n u c l e o s i d e s . S u b s e q u e n t in vitro e x p e r i m e n t s d e m o n s t r a t e d a r e q u i r e m e n t f o r A T P f o r s e m i - c o n s e r v a t i v e synthesis a n d f o r the ability to synthesize b i o l o g i c a l l y active D N A .

381

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FIG. 19. Semiconservative synthesis and transforming activity of DNA synthesized in the presence of ATP in toluene-treated B. subtilis. B. subtilis 168TT was grown at 30° in a synthetic medium containing 1*C-thymine to 7 x 107 cells/ml, harvested, washed, resuspended in phosphate buffer, and agitated for 10 min at 25° with 1 ~/o toluene. Appropriate amounts of stock solutions were added to yield the following reaction mixture: 70 mM K-PO,, pH 7.4, 13 m i Mg2SO,,, 1.3 mM ATP, 33 /zM dGTP, dATP, and dCTP, 30/~M dBUTP, 3 /~m 3H-TTP, 2 mM dithiothreitol. Incubation was for 60 min at 37°. Following lysis using EDTA-lysozyme and sodium dodecylsulfate,CsCl was added, and the solution centrifuged for 72 hr at 35,000 rpm at 25°. Drops were collected into 1 ml SSC. 0.1 ml aliquots were assayed for transforming activity, and the remainder for radioactivity. Transformations were performed by adding the 0.1 ml aliquots to 1 ml competent cells, shaking at 37° for 40 min, and plating on selective plates. A. Radioactivity profile, x - x - - x , aH in vitro label; - - O - - O - - , t*C prelabel. B. Transforming activity, x - - x - - x , ade6; - - O - - O I , leu8; i O - - O - - , met5; , relative amount of DNA. Figure from T. Matsushita, personal communication.

2. L y s e d cell systems As more is learned a b o u t the requirements a n d necessary precautions in the c o n s t r u c t i o n of in vitro D N A replication systems, developments will proceed in the direction of more highly purified, fewer c o m p o n e n t systems. The permeable systems described above result in essentially n o purification. Further, the cells are permeable primarily only to small molecules, although the D N A in the toluene-treated cells is at least partially D N a s e sensitive. Three types of lysed cell in vitro D N A synthesis systems have been developed: the cellophane system (Schaller et al., 1972), the agar system (Smith et al., 1970), a n d the m e m b r a n e systems ( G a n e s a n a n d Lederberg, 1965; K n i p p e r s a n d Str~itling, 1970; Okazaki et aL, 1970).

382

DOUGLAS W. SMITH

In the cellophane system of Schaller et al. (1972), E. cob H560 polA1 endoI- cells are concentrated to about 5 × 101° ceUs/ml, immobilized as a monolayer on a cellophane surface, and gently lysed at 0°C using a lysozyme-EGTA-Brij 58 osmotic shock procedure. The concentrated lysate is dried on the cellophane disc and 50/zl of reaction mixture similar to that shown in Table 3 is added to the disc. The cellophane is similar to dialysis tubing and permits diffusion of small molecules through the disc, but all large molecues are retained. Thus, no purification of the replication apparatus is possible with this system. However, any molecules or aggregates can be added to the system. Characteristics of the in vitro D N A synthesis are similar to those shown in Table 3. D N A TABLE 3 THE AGAR in vitro D N A SYNTHEStS SYSTEM

Typical Reaction Mixture In 0.3 ml: 0.1 M KCI; 0.05 M tris-Cl, pH 7.4; 5 mM MgCI2; 4 mM mercaptoethanol (MCE); 1 . 5 m i ATP;0.01 mM s R N A ; 0.62 mM CdR; 20/~M dCTP, dATP, dGTP, TTP; 0.1 mC//LM dCTP (H a or a-Pa2).

Characteristics of the Incorporation System dCMP incorporated into acid-insoluble material pmoles/10 s cells Complete System Minus TTP Minus dATP, TTP Minus ATP Plus 50/~g pancreatic DNase, 50 units venom phosphodiesterase During incubation After incubation Plus 1.5 m i N E M , - - M C E Plus 0.1 mM pHMB, - - M C E Plus 10 mM K C N Plus 10 m i NaN3 Plus 0.2 m i araCTP

%

31.0 7.5 3.6 16.0

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synthesis proceeds at a decreasing rate for greater than 60 min, at an overall rate and chain elongation rate about 2 0 ~ of the in vivo rate. The in vitro D N A synthesis mimics in vivo D N A replication in many respects. Synthesis is semi-conservative, and treatments which specifically inhibit in vivo D N A replication correspondingly affect the rate of the in vitro synthesis. Synthesis is reduced at the restrictive temperature when lysates of thermosensitive dna mutants are used. The D N A is synthesized in short pieces which are joined together upon prolonged incubation. The major feature of this system is the high concentration of all components. Upon dilution of the reaction mixture, the reaction ceases after 5 to 10 min. Thus, essential soluble molecules must be present at a concentration comparable to their in vivo concentration. The system has been used to monitor the purification of a soluble protein which complements a crude extract of an E. eoli dnaE mutant at the restrictive temperature (NiJsslein et al., 1971). This gene product, indistinguishable from D N A polymerase III (Kornberg and Gefter, 1971), is described in section IIICI. In a second application, single-stranded viral

D N A SYNTHESIS IN PROKARYOTES: REPLICATION

383

× 174 DNA can be converted into the double-stranded RF forms when added to E. coli H560 extracts in the cellophane system. Predominately RF II accumulates, but is slowly converted to the RF I form, in a reaction that requires the E. eoli NAD-dependent DNA ligase (Olivera and Bonhoeffer, 1972). In the agar system, exponentially growing E. cob cells are immobilized in an agarose gel, the gel is fragmented, and the cells are converted into spheroplasts (Smith et al., 1970). A variety of E. cob strains have been used. The spheroplasts are routinely lysed by washing the agar fragments with an appropriate lysing buffer through a nitrocellulose filter. Large and small soluble molecules are thus washed out of the lysed cells, resulting in a semi-purified DNA synthesis system. For example, about 75~ of DNA polymerase I is washed from E. cob polA ÷ cells (Smith, unpublished observation). Further,-it is possible to add large molecules to the system, including aggregates as large as bacteriophage T7. Characteristics of the system are basically similar to those of the permeable systems. A typical reaction mixture, and properties of the reaction, are given in Table 3. ATP stimulates synthesis about two-fold, and is required for semi-conservative synthesis (Fig. 17). Other adenine derivatives, including dATP, will not substitute for ATP, and ADP is inhibitory. K + is optimal at about 0.08 M, and all four dNTPs and Mg + + are required for synthesis. Mn + + can partially substitute for Mg* +, and Ca ++ is inhibitory. Na +, NH, +, and SO, = are inhibitory. Synthesis is inhibited by NEM, pHMB, and araCTP, and partially inhibited by azide, DNP and cyanide. The product DNA and endogenous DNA substrate are DNase-sensitive. The synthesis thus does not proceed in a few remaining intact cells. Synthesis is inhibited by pronase, by detergents such as sarkosyl or SDS, and by disruption with sonication. Treatments which specifically inhibit in vivo DNA replication inhibit the in vitro DNA synthesis, and the in vitro synthesis is reduced at the restrictive temperature in some thermosensitive E. cob dna mutants (Smith, manuscript in preparation). The initial rate of synthesis is close to the in vivo rate, but decreases in time. In early experiments, synthesis stopped after about 5 min (Smith et al., 1970); in further modifications of the system, synthesis will proceed for at least 60 min, although at gradually reduced rates. Synthesis in the absence of ATP ceases after about 5 min. The product DNA synthesized after a short period of time, with dBUTP substituted for TTP in the reaction mixture, is hybrid in density, and is heterogeneous in molecular weight (Smith et ai., 1970). Figure 17 shows the results of the first CsCI gradient performed using this system, executed in July 1969. In the presence of ATP, the newly synthesized DNA is found in two peaks, one at the density of BU-hybrid DNA, and the other at a density slightly heavier than the parental thymine-containing DNA. No hybrid density DNA is synthesized in the absence of ATP. When the DNA is reduced in molecular weight by sonication prior to CsC1 analysis, nearly all of the DNA synthesized in the presence of ATP is found at hybrid density (Smith et al., 1970). The results shown here, done with a polA + strain prior to the availability of the p o l A - strains, are also observed with p o I A - strains (Smith et al., 1970). Semi-conservative synthesis is also observed with r e e A - , reeB-, and recC- strains (Smith, manuscript in preparation). In the hybrid density DNA, most of the BUcontaining DNA is not covalently bound to the parental DNA. However, some (about 10~) appears to be covalently attached during synthesis for 5 min at 37°; the physiological significance of this observation is under study. All membrane in vitro DNA synthesis systems involve gentle lysis of bacterial cells, with subsequent purification of a "membrane" fraction by centrifugation. The rationale for using this procedure to obtain a DNA synthesis system is based on the replicon hypothesis

384

DOUGLAS W. SMITH

(Jacob et al., 1963) and the rapid sedimentation properties of newly synthesized DNA (Ganesan and Lederberg, 1965; Smith and Hanawalt, 1967). Ganesan and Lederberg (1965) observed that most of the DNA polymerizing activity from B. subtilis cells sedimented slowly in sucrose gradients, but that some activity was found in the pellet with the newly synthesized DNA. This pellet was subsequently used to synthesize in vitro DNA which was biologically active. Heavy N is, H2-1abeled B. subtilis DNA was added as a substrate for a first round of in vitro DNA synthesis. Using isolated hybrid DNA obtained from this first round of in vitro DNA synthesis as a DNA substrate for a second round of in vitro DNA synthesis, Ganesan (1968a) was able to obtain some double-stranded DNA which was synthesized totally in vitro and had biological activity in the transformation assay system. Knippers and Str~itling (1970) have obtained a membrane DNA synthesis system by centrifuging gently lysed E. coli H560 polA1 endoI- cells infected with phage ~ × 174 onto a 60~ sucrose pad in a sucrose gradient. The cells were converted to spheroplasts by lysozyme-EGTA treatment, and lysed with Brij 58 and Mg + +. Fractions from the top of the sucrose pad constitute the membrane system. Characteristics of the synthesis are nearly the same as observed with the agar system (see Table 3). DNA polymerase I, from a p o l A ÷ control experiment, was found in the upper part of the sucrose gradient. Analysis of the DNA synthesized in vitro using membranes from uninfected cells showed that most of the product DNA had a low molecular weight. Using ~ × 174-infected cells, the phage DNA synthesized in vitro was predominantly RFII. CsC1 gradient analysis of this RFII DNA, obtained from an in vitro reaction using dBUTP instead of TTP, is shown in Fig. 18. Most of the parental P32-1abeled phage DNA is found in the light DNA peak, although some has been converted into hybrid density (heavy-light DNA). The DNA synthesized in vitro (H3-1abeled) is mostly of hybrid density, with a little at light density. However, about 25~ is fully BU-labeled, suggesting that some DNA has been totally synthesized in vitro. Of even greater significance, this DNA is biologically active, as tested using the spheroplast assay system. In further studies, Str~itling and Knippers (1971a) have examined the composition of the material in their rapidly sedimenting replication complex. Most of the cell wall structures, cell membrane, and bacterial chromosome, with 20~o of the total cell protein, are found in this fraction. Selective solubilization and disruption of the complex with detergents, lipase enzymes, and sonication showed that the DNA synthesizing activity was attached to the bacterial chromosome rather than to some other component. The product DNA has been further analysed (Str/itling and Knippers, 1971b). After deproteinization, part sediments in sucrose gradients with the parental DNA and part sediments more slowly (about 15S). CsC1 gradient analysis of density-labeled product DNA showed that most of the product DNA is noncovalently attached to the parental DNA, with the exception of one fraction of product DNA which appears to be covalently attached to the parental DNA. In similar studies, Okazaki et al. (1970) used a pellet fraction of a lysozyme-EDTA-Brij 58 lysate of E. coli or T4-infected E. coli. The DNA synthesized in vitro had properties similar to those of newly synthesized DNA in vivo. Infection with gene 43 or gene 44 (see Table 1) T4 mutants resulted in rapid disappearance of DNA synthesizing activity, as expected. Linney and Hayashi (1971) have obtained a slowly sedimenting DNA synthesizing activity from ~× 174-infected E. coli H560 p o l A l endoI- cells lysed using a lysozymeEGTA-Brij procedure. DNA-DNA hybridization showed that the product DNA hybridized only with ~× 174 RF DNA, but not with ~× 174 viral DNA or with E. coli DNA, suggesting that the single-stranded ff x 174 DNA synthesizing activity had been isolated.

D N A SYNTHESISIN PROKARYOTES: REPLICATION

385

The in vitro DNA synthesis ability of toluene-treated E. cob polA4 rec + and E. cob p o l A 4 reeB21 cells has been compared with the DNA synthesizing ability of lysozyme-Brij lysates from these cells (Bazill et al., 1971). The ATP-stimulated synthesis was reduced about 4-5 times in the lysates of the reeB21 cells compared with the rec ÷ cells, whereas no significant difference was noted when the cells were toluene-treated. Since the recB gene product has an ATP-stimulated nuclease activity, these authors concluded that the synthesis observed in lysates or in membrane DNA synthesis systems is an artifact due to this nuclease activity. Miller and Kozinski (1970b) immobilized DNA-protein complexes obtained from T4infected E. eoli on glass fiber filters. These complexes could incorporate radioactive DNA precursors into both E. coli and phage DNA. The product appeared to be covalently linked to the DNA substrate. E. Mechanisms and Models 1. Discontinuous synthesis

The Okazaki fragments, observed only with newly synthesized DNA, were discussed in section IIIA3. Their discovery provided a rationalization to the following basic problem (Mitra and Kornberg, 1966; Richardson, 1969; Lark, 1969): in the apparent absence of a DNA polymerase which can use a DNA 5'-end as primer, what is the mechanism for chain elongation of the daughter DNA strand whose 5'-end is at the replication fork ? A discontinuous synthesis model in which chain elongation proceeds on this daughter strand in the opposite direction for short distances, was first proposed by Kornberg and co-workers (Mitra and Kornberg, 1966; Kornberg, 1969 review). A polymerase with the in vitro properties of DNA polymerase I is the only polymerase required in the model. In a more general analysis, but assuming no DNA structural changes ahead of the replication fork, three types of DNA structures may be envisioned (Okazaki et al., 1968a, b), as shown in Fig. 20. Either one (Fig. 20A) or both (Fig. 20B) of the daughter DNA strands might be synthesized discontinuously. In addition to these two possibilities, either one or both of the parental DNA strands could suffer temporary nicks, or extension to gaps, behind the replication fork during replication (Fig. 20C). Many possibilities regarding mode of generation and temporal appearance of these structures exist. Analysis of the structures found in newly replicated DNA would appear to limit some of these possibilities. Nearly all of the most newly replicated DNA (70~ or more) is found as short fragments in B. xubtilis, in SPP-1 infected B. subtilis (Polsinelli et aL, 1969), E. coli, and T4-infected E. coli (Okazaki et al., 1968a, b; Sadowski et al., 1968; Yudelevich et al., 1968), although this has not always been observed (Iyer and Lark, 1970). Several workers (Richardson et al., 1968; Sugimoto et al., 1968; Newman and Hanawalt, 1968a, b; Okazaki et al., 1968b; Kozinski, 1968; Hosoda and Mathews, 1968) have also shown that infection of E. coli with ligasedeficient T4 under non-permissive conditions leads to the accumulation of low molecular weight T4 DNA fragments (about 10S); return to permissive conditions results in conversion of these fragments to high molecular weight. However, interpretation of these observations is complex since introduction of an rII mutation into the same phage suppresses the above effects of gene 30 mutants and restores DNA synthesis (Berger and Kozinski, 1969; Karam, 1969; Chan et al., 1970). Infection of E. coli with gene 30 T4 mutants in the presence of chloramphenicol leads to a similar suppressive effect (Berger and Kozinski, 1969; Iwatsuki and Okazaki, 1970; Hosoda and Mathews, 1971). These results are consistent with a hypothesis that the rII gene controls an endonuclease activity; the T4 ligase activity during normal

386

DOUGLAS W. SMITH A

C

FIO. 20. Models for the possible structure in the D N A replication fork. A. Discontinuous synthesis in one of the daughter D N A strands. B. Discontinuous synthesis in both of the daughter D N A strands. C. Discontinuous synthesis in both of the daughter D N A strands, with nicks introduced into both parental D N A strands. Such models do not consider possible structural changes and biochemical events ahead of the replication fork (for example, see Fig. 22). Adapted from Okazaki e t al. (1968b).

infection would limit the extent of the nuclease activity. However, the rlI gene product could also be an inhibitor specific for the host E. coli DNA ligase. Chloramphenicol or rlI suppression does not occur when T4 ligase mutants infect ligase-deficient E. coli mutants, consistent with either of the above interpretations (Gellert and Bullock, 1970). The existence of newly synthesized DNA as short fragments suggests that chain elongation at both termini of the daughter strands may proceed in a discontinuous manner (Okazaki et al., 1968a, b). Thus, structures such as shown in Figs. 20B and 20C are favored. Further, when the DNA is isolated using non-denaturing methods, the short 10S fragments are still found, suggesting that discontinuities in the parental strands exist. However, this may be an isolation artifact due to unusual sensitivity of the replicating DNA to fragmentation and/or denaturation, either by shear, by local environment, or by nuclease activity. Hybridization experiments indicate that Okazaki fragments are synthesized from both parental DNA strands for E. coil infected with phages T4, T7, and lambda (Okazaki and Okazaki, 1969; Sugimoto et al., 1969; Ginsberg and Hurwitz, 1970, Tomizawa and Ogawa, 1968; KainumaKuroda and Okazaki, pers. comm.) but probably only from one parental strand for B. subtilis and for P2-infected E. coli (Kainuma-Kuroda and Okazaki, pers. comm.). Since DNA replication is bidirectional for E. coil (Masters and Broda, 1971 ; Bird et al., 1971), for

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lambda (Schn6s and Inman, 1970), and for T7 (Wolfson et al., 1972), but appears to be unidirectional for B. subtilis (Sueoka, 1967) and for P2 (Schn6s and Inman, 1971), interpretation of the hybridization results is difficult. Using a double-labeling method with very short pulse times at reduced temperatures and selective nuclease digestion of isolated fragments, Okazaki and Okazaki (1969) have shown that synthesis proceeds exclusively in a 5'- to T-direction in T4-infected E. coil, consistent with the above mechanisms. 2. The rolling circle model

A more specific discontinuous synthesis model, with features of the above as well as for initiation of DNA replication, is "the rolling circle" model (Gilbert and Dressier, 1968; Eisen et al., 1968; Kiger and Sinsheimer, 1969a). Properties and predictions of this model have been reviewed in detail (Gilbert and Dressier, 1968; Richardson, 1969; Lark, 1969; Goulian, 1971); only the basic features will be considered here. A putative nickase attacks the non-replicating DNA molecule, assumed to be a closed double-stranded helix, at a specific site (Fig. 21A). The 5'-end of the single-strand break is transferred to a "site" in the cell, for example, on the cell membrane (Fig. 21B). The other closed parental DNA strand serves as a template for chain elongation at the T-OH-end of the opened parental D N A strand, which serves as primer (Fig. 21C). The primer parental strand continues to be "peeled off" until a second "initiation" site is exposed (Fig. 21E), for example, a specific binding site for a small primer oligodeoxynucleotide. Synthesis then proceeds in the reverse direction, using the open parental DNA strand as template (Fig. 21E). Any of the known polymerase enzymes could catalyse the above synthesis, and DNA ligase could join the segments of newly synthesized DNA. In agreement with the rolling circle model, DNA structures which sediment rapidly and, in some cases, are longer than unit viral DNA in length have been found in E. eoli cells infected with phages T4 (Frankel et al., 1968; Werner, 1968; Shah and Berger, 1971; Shalitin and Naot, 1971), T5 (Pinkerton, 1968), T7 (Hausmann, 1968; Kelly and Thomas, 1969), P22 (Botstein and Levine, 1968), lambda (Weissbach et al., 1968; Young and Sinsheimer, 1968; Kiger and Sinsheimer, 1969a, b), and 4, × 174 (Dressier and Denhardt, 1968; Knippers et al., 1969b; Dressier and Wolfson, 1970; Gilbert and Dressier, 1968). However, their role in DNA replication remains unclear. Upon infection, the parental phage lambda 1 strand was found to be in monomer circles, whereas tb.e parental r strand was present in linear forms 2 to 5 times unit length (Ihler and Kawai, 1971), consistent with asymmetric replication and the rolling circle model. During ff × 174 infection, one of the parental phage DNA strands appears to be noncircular and longer than unit length. This open strand has been reported to be the viral, or + , strand by some groups (Gilbert and Dressier, 1968; Dressier and Wolfson, 1970), and the complementary, or --, strand by others (Knippers et al., 1969b). Further, during 4, × 174 single-strand DNA synthesis, doublestranded circles with single-stranded tails have been observed in electron micrographs (Knippers et al., 1969a; Dressier, 1970). A modification of the rolling circle model has been proposed to account for bidirectional replication; this has been termed the "double rolling circle" model (Dressier and Wolfson, 1971). In essence, following the establishment of one replication fork by the mechanism described above, the other closed parental D N A strand suffers a single-strand break, the 5'-end so exposed is peeled off and transferred to a site, and chain elongation proceeds at the exposed 3'-OH end. Thus, two replication forks, moving in opposite directions, are generated. As an alternative, the two 5'-ends might anneal with each other rather than being

388

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FIG. 21. The rolling circle model for DNA replication. An endonucleolytic single-strand break is introduced into one strand of the doublestranded DNA substrate at a specific site (O). The 5'-end is transferred to some intracellular site (D). The exposed 3'-OH of the ÷ strand serves as a primer and the other circular parental DNA strand serves as a template for extension of the ÷ strand from the 3'-OH end. In the knife and fork modification, the polymerizing enzyme loops back and uses the + strand as template. The process begins again when an endonuclease introduces a nick into the newly-synthesized DNA at the replication fork, leading to E. When a second initiation site (O) becomes exposed, DNA synthesis proceeds from this second site using the + strand as template. A deoxyribonucleotide oligomer could possibly serve as primer in this initiation of synthesis (Goulian, 1968b). A possible structural DNA intermediate of the knife and fork version, after three rounds of replication. A possible structural DNA intermediate of the second initiation site version, after three rounds of replication. Adapted from Richardson (1969) and Goulian (1971).

transferred to some site. Using the second initiation site possibility described above, these two parental strands would be symmetrically replicated via a Cairns-type replication intermediate, with two replication forks progressing in opposite directions. Note, however, that replication w o u l d still proceed in a discontinuous, albeit symmetrical, m a n n e r . As a n alternative to the second i n i t i a t i o n site in the single rolling circle model, the polymerase catalysing e l o n g a t i o n o f the 3'-end of the nicked parental D N A strand might at

DNA SYN'rnESISIN PROKARYOTES:REPLICATION

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some point shift from the other closed parental DNA strand as template to the open parental strand (see Fig. 21D). Such a shift in templates was proposed by Schildkraut, Richardson, and Kornberg (1964) and Kornberg (1969) as a possible mechanism for the generation of a branched DNA product observed in the extensive synthesis reaction by DNA polymerase I (see Fig. 14E). This idea was specifically suggested by Guild (1968) as a possibility for DNA replication, and has been combined with the rolling circle model to yield the "knife and fork" model (Richardson, 1969; Lark, 1969; Kornberg, 1969; see Fig. 21D, F). To synthesize a new segment, a nickase would insert a single-strand break into the newly synthesized DNA at the replication fork. Following the unwinding of the bihelical parental DNA ahead of this single-strand break, synthesis would proceed as before, using the 3'-OH end of the progeny DNA as primer and the unwound single-strand region of the parental DNA as template. Because of its complementary nature, this model predicts that the most newly synthesized DNA, prior to insertion of the single-strand break at the fork, should rapidly reanneal following melting. Rapid renaturation has been observed for pulse-labeled DNA in E. coli (Pauling and Harem, 1969a, b) and in T7-infected E. coli(Barzilai andThomas, 1970; Morgan, 1970). However, the presence of the small E. coli 15 plasmid DNA (Cozzarelli et al., 1968)in the E. coli TAU-bar strain used in these experiments (Pauling and Hamm, 1969a, b) introduces some uncertainty in the interpretation of these results. 3. The pre-fork synthesis model Another discontinuous model for DNA replication, symmetrical with respect to the two parental DNA strands, has been proposed by HaskeU and Davern (1969). The model, envisaging chain elongation ahead of the replication fork, is called the "pre-fork" synthesis model, and is shown diagrammatically in Fig. 22. A specific endonuclease recognizes multiple initiation sites on the two parental DNA strands (O), and introduces nicks with 3'-OH ends (Fig. 22A). These ends serve as primer DNA for chain elongation, using the other parental DNA strand as template and displacing the 5'-phosphoryl end of the nicked parental DNA (Fig. 22B). Chain elongation is thus basically a repair synthesis (see Fig. 14E), consistent with the in vitro reactions of known DNA polymerases. The specific endonuclease again recognizes the multiple initiation sites (O), and introduces nicks which now separate the newly synthesized DNA from the parental primer DNA (Fig. 22C). The displaced 5'phosphoryl ends of the parental DNA are free to return to the initiation sites, where they are rejoined to the 3'-OH ends of the parental DNA by a DNA ligase activity (Fig. 22D: []). The nascent daughter strands continue to elongate, generating a four-stranded intermediate with partial double-stranded properties immediately ahead of the replication fork (Fig. 22E). This chain elongation, similar to the repair synthesis of Fig. 14B, continues until the "gaps" are filled, with subsequent fusion of the daughter DNA pieces by DNA ligase (Fig. 22F:11). The pre-fork model has many attractive features. Introduction of transient nicks into the parental DNA readily solves the so-called unwinding problem (Levinthal and Crane, 1956) and obviates the need for a swivel (Cairns, 1963b). The replicating DNA structures are possibly unstable, providing a need for a structural intracellular site. These DNA structures would lead to small pieces upon isolation, perhaps even at neutral pH, as well as to the partial single-stranded nature of newly replicated DNA (Oishi, 1968a, b). Because of the inherent symmetry of the model, the small pieces would be expected to hybridize equally well with both parental DNA strands. The reactions involved can be catalysed by enzymes such as those already isolated and studied. With synthesis proceeding simultaneously at

390

DOUGLASW. SMITH

several small DNA pieces, the overall rate of replication fork movement would be correspondingly higher than the rate of elongation of the individual chains. Thus, the relatively slow rate of synthesis observed in vitro with isolated DNA polymerases could be rationalized in this model. The model makes several testable predictions. One, an endonuclease specific for selected sites on a given DNA duplex should exist. The rolling circle model also makes this prediction. No known E. coli endonuclease exhibits these properties, although it is possible the phage T7-induced endonuclease II (Center, 1972) is such an enzyme. Two, the most newly

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FIG. 22. The pre-fork synthesis model for DNA replication: Haskell and Davern (1969). A. Nicks generated by an endonuclease, yielding Y-OH primer DNA: @. B. D N A synthesis, displacing the parental D N A (see Fig. 14A). C. Endonuclease separates newly synthesized D N A from parental primer DNA. D. Ligase rejoins the transiently nicked parental DNA strands : [D. E. Generation of a four-stranded intermediate, with chain elongation of the newly synthesized D N A daughter strands. F. Fusion of the daughter D N A pieces by ligase. Sites for endonuclease nick in parental DNA: 0 . These same sites after the parental D N A strands are rejoined: 71. New endonuclease sites in the daughter D N A for the next round of replication: II. Parental D N A shown by the light solid line, newly synthesized daughter D N A by the heavy solid line.

synthesized DNA should be found covalently attached to parental DNA. Some evidence for this exists from in vitro studies with the membrane system (Str~itling and Knippers, 1971b), the ether-treated cell system (Geider and Hoffmann-Berling, 1971), and possibly with the agar system Smith, manuscript in preparation). Three, a four-stranded intermediate should be found in the DNA near the replication fork. Fourth, the newly synthesized D N A should hybridize equally well with both parental DNA strands. A variation of the pre-fork model has been proposed by Denhardt (1972). The central feature of this model is the postulate that "primer" proteins called rr proteins exist. These proteins interact with specific multiple initiation sites on the parental D N A strands during replication, effecting initiation of synthesis of short pieces of nascent DNA. The nascent DNA is not covalently attached to parental DNA at any time. An endonuclease which would interact with the 7r proteins would nick the parental DNA, permitting DNA unwinding without a swivel. The parental DNA would be rejoined when the ~rprotein and nuclease dissociate from the D N A . ATP would be required for replication both as an allosteric cofactor for the nuclease---rr protein interaction and as an energy source for rejoining of the nicked parental DNA strands. Although no ~r proteins have been identified as such, several possibilities exist. The omega protein (Wang, 1971 ; see section IIIC5) has many of the properties suggested for the ~rprotein-nuclease complex. ATP-dependent nucleases exist (Oishi, 1970; see section IIIC3). The supposed cis-acting gene products (see section IIIB3) of ff x 174 gene A (Hutchison, 1969), phage P2 gene A (Lindahl, 1970), and the phage lambda class C mutants (Rambach and

D N A SYNTHESISIN PROKARYOTES" REPLICATION

391

A

FIG. 23. Possible DNA replication intermediates. A. Replicating DNA from phage T4-infected E. coli. Numerous reinitiations are observed within the replicative loop. Figure from Delius et al. (1971). B. Closed circular mitochondrial DNA from the LA9 cell line prepared for electron microscopy using the formamide technique. Displacement loops are shown by arrows. A single-stranded ¢ x 174 is shown in the insert. Figure taken from Kasamatsu et al. (1971). C. Replicating colicinogenic factor E1 DNA. Three examples of supposedly replicating supercoiled (form I) DNA molecules are shown, at different stages of replication. Figure taken from Fuke and lnselburg (1972). Brachet, 1971) could possibly function as ~r proteins. The phage lambda O and P gene products, p r o b a b l y nucleases, the T7-induced endonuclease II, and the T4-induced endonuclease I I (see section IIIC3) are possibilities for the proposed endonuclease. F o r m I superhelical mitochondrial D N A species with a short polydeoxynucleotide h y d r o g e n - b o n d e d to the mitochondrial D N A strands o f lower b u o y a n t density have recently been observed in electron micrographs (Kasamatsu e t al., 1971); an example is shown in Fig. 23B. The short three-stranded region causes the heavy D N A strand to loop out,

392

DOUGLAS W. SMITH

forming a "displacement loop", or "D-loop". D-loops are formed at a unique site on the mitochondrial DNA molecule, and have various sizes, up to unit length, for the DNA molecule. These are thought to be replication intermediates in the first stage of progeny molecule formation. These findings support the hypothesis that initiation of DNA replication can occur without covalent attachment of newly synthesized DNA to parental DNA. However, it is possible that an endonuclease breaks the phosphodiester bond joining parental and progeny DNA very shortly after initiation, before the joining can be observed. Two other examples of supposedly replicating intermediate DNA structures are shown in Fig. 23. In Fig. 23A, multiple reinitiations are observed within the replication loop of a replicating phage T4 molecule. Note the asymmetry in replication. In Fig. 23C, a type of replicating form I supercoil DNA molecule is shown. Col factor E1 DNA molecules at supposed different stages of replication are shown. DNA replication is immediately inhibited by cyanide or azide, before the presumed DNA precursor pools have been depleted (Cairns and Denhardt, 1968; Olivera and Lundquist, 1971), suggesting that the DNA replication process itself is energy-dependent. The in vitro DNA synthesis systems require ATP for semi-conservative synthesis (see section IIID). Thus, ATP may be used directly in DNA replication, and the cyanide inhibition may be due to inhibition of ATP biosynthesis. Such an interpretation is consistent with the above model. An alternative interpretation is proposed by Olivera and Lundquist (1971). In the presence of cyanide, a small amount of DNA synthesis occurs, but this DNA contains many singlestrand breaks. Further, cyanide promotes reduction of NAD to N A D H suggesting that DNA ligase inhibition is the mechanism for DNA synthesis inhibition by cyanide. IV. CONCLUSIONS This article has attempted to survey the basic known features, some recent exciting developments, and current ideas and models concerning DNA replication in prokaryotes. The biochemical reactions and control mechanisms for the process of DNA replication remain poorly understood. The number of features "known with certainty" is remarkably small. Even these are subject to scrutiny and change, as witness the recent downfall of a unidirectional mode of replication in some prokaryotic organisms. A genetic analysis of DNA replication in bacteria is very recent and has only just begun. For the bacteriophage, such analysis is somewhat more advanced, but the most important discoveries probably remain for the future. Some enzymes possibly relevant to DNA replication have been extensively studied. Based on the in vitro properties of these enzymes, several models for DNA replication have been proposed and experimentally tested. However, the role of these enzymes in DNA replication and the relevance of the properties of the enzymes as observed in vitro to the enzyme properties in vivo remain unclear. Two recent developments have rejuvenated interest in this biological problem, and hold promise for rapid progress in the near future. These include the discovery of the E. coli mutants deficient in DNA polymerase I, and the development of in vitro DNA synthesis systems which closely mimic certain features of DNA replication. Isolation of E. coli W31 l0 polA1, and its subsequent study, has shown that DNA polymerase I is not essential for DNA replication. The use of such mutants to eliminate the high background of nonessential polymerizing activity due to DNA polymerase I has permitted biochemical studies otherwise not possible or technically very difficult. The in vitro DNA synthesis systems, with their emphasis on integrity of the replication apparatus, promise to provide a systematic

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increase in k n o w l e d g e a b o u t the structure a n d f u n c t i o n o f the D N A r e p l i c a t i o n a p p a r a t u s . The present time r e g a r d i n g this process a p p e a r s similar to the p e r i o d in the early a n d m i d 1950s r e g a r d i n g p r o t e i n biosynthesis. A s the relevant features o f the replication a p p a r a t u s unfold, i m p r o v e m e n t s in the in vitro systems will a p p e a r . This c o u p l e d with the genetic analysis, b o t h in b a c t e r i a a n d in their b a c t e r i o p h a g e , p r o m i s e s a r a p i d increase in o u r p r e s e n t very limited k n o w l e d g e o f the process o f D N A replication.

ACKNOWLEDGEMENTS The a u t h o r wishes to t h a n k the m a n y w o r k e r s who have p r o v i d e d p r e p r i n t s o f their recent research, those w h o generously p r o v i d e d m i c r o g r a p h s for this article, his colleagues at U C S D a n d elsewhere, p a r t i c u l a r l y Drs. E. P. G e i d u s c h e k , M. G o u l i a n , P. C. H a n a w a l t , D. R. Helinski, a n d S. Howell, for e n c o u r a g e m e n t , discussion, a n d helpful i n f o r m a t i o n a n d criticism, a n d Dr. W . H e l f m a n , Dr. S. Howell, Mr. O. R y d e r , a n d Mr. P. Schmitt for detailed r e a d i n g a n d criticism o f the text. The a u t h o r ' s w o r k was s u p p o r t e d b y a research grant, E-625, f r o m the A m e r i c a n C a n c e r Society. REFERENCES AARONSON,S. A., PARKS,W. P., SCOLNICK~E. M. and TODARO,G. J. (1971) Proc. Nat. Acad. Sci. U.S. 68, 920. ABE, M. and TOMIZAWA,J. (1967) Proe. Natl. Acad. Sci. U.S. 58, 1911. ACKERMAN,W. W., MURPHY, W. H., MILLER,B. A., KURTZ,H. and BARKER,S. T. (1971) Biochem. Biophys. Res. Comm. 42, 723. ADES~K, M. and LEVINTHAL,C. (1970)J. Mol. Biol. 48, 187. ADLER,H. I., FISHER,W. D., COHEN,A. and HARDIGREE,A. A. (1967) Proc. Nat. Acad. Sci. U.S. 57, 321. ADLER, H. I., FISHER,W. D. and HARDIGREE,A. A. (1969) Ann. N. Y. Acad. Sci. 31, 1059. ALnERTS,B. M. (1970) Fed. Proc. 29, 1154. ALBERVS,B. M. (1971) In Nucleic Acid-Protein Interactions p. 128. (Ribbons, Wessner, and Scholtz, eds.) North Holland, Amsterdam. ALBERTS,B. M., AMODIO,F. J., JENKINS,M., CUTMANN,E. D. and FERRIS,F. L. (1968) Cold Spring Harbor Syrup. Quant. Biol. 33, 289. ALBERTS,B. M. and FREY,L. (1970) Nature, 227, 1313. ALBERTS,B. M., FREY, L. and DELIUS,H. (1972) J. Mol. Biol. 68, 139. ALLEN, E. F., ALBRECHT,I. and DRAKE,J. W. (1970) Genetics 65, 187. ALTENBURG,B. C. and SUIT,J. C. (1970) J. BacterioL 103, 227. ALTMAN,S. and LERMAN,L. S. (1970a) J. Mol. Biol. 50, 235. ALTMAN,S. and LERMAN,L. S. (1970b) J. Mol. Biol. 50, 263. ALTMAN,S. and MESELSON,M. (1970) Proc. Nat..4cad. Sci. U.S. 66, 716. ANAL M., HIRAHASHI,T. and TAKAGI,Y. (1970a) J. Biol. Chem. 245, 767. ANAl, M., HIRAHASHI,T., YAMANAKA,M. and TAKAGI,Y. (1970b)J. Biol. Chem. 245, 775. ANAI,M. and TAKAGI,Y. (1971) J. Biol. Chem. 246, 6389. ANDO, T., TAKAGI,J., KOSAWA,T. and IKEDA,Y. (1969) J. Biochem. Japan 66, 1. ANDO, T., TAKAGI,J., KOSAWA,T. and IKEDA,Y. (1970) J. Biochem. Japan 67, 497. ANRAKU,N. and LANDMAN,O. E. (1968) J. Bacteriol. 95, 1813. APOSHtAN,H. V. (1968) In Molecular Basis o f Virology p. 497 (Fraenkel-Conrat, H., ed.) Reinhold, New York. APOSHIAN,H. V. and KORNBERG,A. (1962)./. Biol. Chem. 237, 519. ARBER,W. and LINN, S. (1969) Ann. Rev. Biochem. 38, 467. ATKINSON,M. R., DEUTSCHER,M. P., KORNBERG,A., RUSSELL,A. F. and MOFFATT,J. G. (1969) Biochem. 8, 4897. BALDWIN,R. L. and SHOOTER,E. M. (1963) J. Mol. Biol. 7, 511. BALDY,M. W. (1968) Cold Spring Harbor Syrup. Quant. Biol. 33, 333. BALTIMORE,O. (1970) Nature, 226, 1209. BALTIMORE,O. and SMOLER,D. (1971) Proc. Nat. Acad. Sci. U.S. 68, 1507. BARnOUR,S. D. (1967) J. Mol. Biol. 28, 373. BARaOUR,S. D. and CLARK,A. J. (1970) Proc. Nat. Acad. Sci. U.S. 65, 955. BARnOUR, S. D., NAGAISHI,H., TEMPLIN,A. and CLARK,A. J. (1970) Proc. Nat. Acad. Sci. U.S. 67, 128.

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