Reconstitution of Mammalian DNA Replication

Reconstitution of Mammalian DNA RepIicat ion ROBERT A. BAMBARA LIN HUANG

AND

Department of Biochemistry and Cancer Center Uniuersity of Rochester School of Medicine and Dentistry Rochester, New York 14642

I. Initiation at Replication Origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Priming Reactions That Initiate DNA Replication . . . . . . . . . . . . . . . . . .

111. Mechanisms of Leading- and Lagging-strand DNA Synthesis . . . . . . . . IV. Completion of Lagging-strand Synthesis .......................... V. Regulation of Replication Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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This review is focused on efforts to perform the series of reactions necessary to carry out mammalian chromosomal DNA replication using purified enzymes in uitro. This work fits into a larger framework of genetic and cell biological experiments, using prokaryotes, viruses, and eukaryotes, that must be discussed in parallel with the work emphasized here. What follows is also meant to complement a number of excellent reviews on eukaryotic DNA replication (1-12). The particular features of DNA replication that we will emphasize include initiation at replication origins, and components and propagation of the replication fork.

1. Initiation at Replication Origins

A. The SV40 System Current approaches to the reconstitution of the mammalian DNA replication fork derive from the work of Li and Kelly (13).They were able to carry out replication of SV401 origin-containing plasmids in monkey-cell extracts, Abbreviations: ARS, autonomously replicating sequence; ABF, ARS binding factor; ORC, origin recognition complex; OBF, origin binding factor; CBF, core sequence binding factor; SSB, single-stranded DNA binding protein; RP-A, replication protein A; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; SV40, simian virus 40; HIV-RT, human immunodeficiency virus-1 reverse transcriptase. Progress in Nucleic Acid Research and Molecular Biology, Vol. 51

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which led to closed circular form I products. This required use of SV40infected cell extracts or supplementation with purified T antigen. The accomplishment of complete synthesis suggested that all functions necessary for the replication process not directed by T antigen are carried out by cellular proteins. Furthermore, the extract system provided a means to purify these cellular proteins by complementation. Shortly thereafter, three groups began the identification of these replication proteins in human cells

(13-1 6). DNA replication begins when T antigen recognizes and binds SV40's origin of replication. T antigen binds at positions designated site I and site 11 (17) (Fig. 1). Although T antigen function is unique to SV40, the details of its action are important because it probably has common features with proteins that initiate DNA replication at all eukaryotic replication origins. Significant structural features of the SV40 origin have been determined by genetic analysis and site-directed mutagenesis (18).The minimal or core origin contains a 64-bp viral genome sequence (Fig. 1).This core origin suffices to support the initiation of viral DNA replication both in uiuo and in uitro. The 64-bp sequence has three domains. The center domain is the SV40 T-antigen recognition and binding site II, which contains four inverted repeats of the sequence GAGGC in 27 bp (17, 19-25). On the side of T-antigen binding domain site 11, in the direction of early transcription, is a 17-bp imperfect palindrome sequence (Fig. I), which is conserved in other papovaviruses (18). On the side of site I1 in the direction of lute transcription is a 20-bp (A.T)-rich domain that serves as a DNA bending center (26).The T-antigen binding-site I is not included in the minimal origin, but rather just adjacent to the palindrome domain on its early side. Nevertheless, binding of T antigen to site I results in a major increase in the efficiency of initiation of replication both in uiuo and in uitro (27-31). From electron microscopy, ATP not only stabilizes binding of T antigen to the core origin, but also allows it to form a unique structure called the double hexamer (25, 32-34). After the double hexamer assembles, the T antigen carries out an unwinding process (35-37). T antigen initiates unwinding by melting an 8-bp region in the palindrome domain of the SV40 core origin, and concurrently induces a conformational change in the (A.T)rich domain (38, 39). The discovery that T antigen has an intrinsic 3'-to-5' helicase activity (40-42) added to our understanding of the role of T antigen in the initiation of SV40 DNA replication. T antigen is capable of continually unwinding DNA in the presence of RP-A, which stabilizes the melted single-strand region, and topoisomerase I, which relieves positive supercoils in the circular molecule (26,36,37,43).The unwound (U) protein-DNA complex allows initiation of synthesis by DNA polymerase dprimase (44).

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A

FIG. 1. Initiation at the SV40 replication origin.

B. Initiation in Yeast and Mammals In Saccharomyces cerevisiae, replication initiates at cis-acting elements, called autonomously replicating sequences (ARS) (45-48). Their position and activity have been assessed by two-dimensional gel electrophoretic mapping techniques (49-50). The ARS contains an 11-bp (A.T)-rich sequence located within the A element. On its 3’ side is an element of more diverse sequence about 80 nucleotides long and designated the B element. The B element has been called the DNA unwinding element because it readily melts (48,51).It

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has recently been subdivided into three distinct elements, B1, B2, and B3 (52, 53). A six-subunit protein named the origin recognition complex (ORC) binds the A and B1 elements (54).Interaction of the ORC with ARS DNA causes a periodicity of DNase I hypersensitive sites in the A and B elements both in oitro (54)and in permeabilized cells (55). The B3 element (52, 53) need not be located 3’ to the A element to be functional. In ARS121, it is at the 5’ end of the A element, where it can function as a DNA replication enhancer in an orientation- and distanceindependent manner (56).The B3 element binds the OBFlABFl protein (53, 56), which has been purified independently in several laboratories as an ARS binding protein (57-63). A specific and stable multiprotein complex has been assembled stepwise in oitro at the ARS121 origin by Eisenberg and colleagues (63),and analyzed by gel-shift assay. The first step involves ATP-independent binding of ABF-1 and a factor designated OBF-2. The next step is the ATP-dependent binding of the protein designated CBF to the essential core sequence. This suggests that CBF is a component of the ORC. Yeast origin structure, hypotheses concerning origin function, and the ORC have been recently reviewed (7, 64, 65). Animal-cell replication origins are distributed throughout regions called “initiation zones” of several hlobasepairs (kbp) or larger (66). Little is known about the essential features of animal cell origins because of the complexity of direct genetic manipulations in their large chromosomes and lack of suitable assays for origin position and activity (67-69). Schizosucchuromyces pombe also displays replication at initiation zones (70), making it an interesting model for mammalian initiation. Recent results with s. pombe suggest that the 4-kbp zone in the uru4 origin region consists of multiple mutually interfering origins (71). This is consistent with the observation of clusters of origin sequence elements in initiation zones of chromosomes from many eukaryotes (72).

II. Priming Reactions That Initiate DNA Replication In the middle 1970s, several groups reported that the nascent DNA chains synthesized from normal or virus-infected cells contain transient, covalently linked, 5’-terminal oligoribonucleotides about 10 residues long, and of varying sequences (73-76). They were designated initiator RNAs (73). Synthesis of R N A that can be used to initiate DNA synthesis requires the action of a primase. Lehman and colleagues first described the co-

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purification of primase activity with the major species of polymerase a from Drosophila melanogaster embryos (77-79). The immunological evidence for a tight association of primase activity with a partially purified polymerase a from murine Ehrlich ascites cells was provided by Yagura et al. (80, 81). Although the primases obtained from various eukaryotic cells have different structures, their hnction is well conserved (reviewed in 1).The initiator RNA synthesized is in the range of 6 to 14 nucleotides long if there is concomitant DNA synthesis, but could be 24 to 36 nucleotides long if DNA synthesis is not allowed (82, 83). Initiation is usually with rA, but the RNADNA junction sequence is not specific. The primase activity of human polymerase a has been characterized in the presence of specific and complete monoclonal antibody inhibition of the DNA polymerase active site. In the presence of physiological concentrations of NTPs, but dNTPs between 0.08 and 0.8 pM, the primase made a product consisting of strictly alternating tracts of RNA and DNA about 12 nucleotides long (83). These conditions reveal a unique alternating synthesis mechanism of the primase. At higher concentrations of dNTPs, the first segment of RNA was made, followed by a variable-length segment of DNA. The authors postulated that at physiological dNTP concentration, the enzyme becomes stabilized in the deoxy-polymerizing mode &er synthesis of'the first tract of RNA. This w a s presumed to be the product transferred to the polymerase active site for further elongation. A unique feature of primase is its low fidelity. Calf thymus primase will misincorporate NTPs at a frequency as high as 1/200 on homopolymeric templates (84). She& and Kuchta showed that primase readily misincorporates nucleotides in the central and 3' regions of the RNA primer, but accurately incorporates the first two nucleotides (85). After synthesis of a primer on single-stranded DNA, the newly generated primer-template is transferred intramolecularly to the active site for DNA synthesis on the large subunit of polymerase a,for subsequent deoxynucleotide addition (86, 87). This coordination of RNA and then DNA synthesis occurs each time a primer is initiated. The signal that governs the switch from RNA to DNA synthesis is intrinsic in the primase mechanism, and is generated by ambient dNTPs (83). Interestingly, polymerase a will efficiently elongate primase-generated primers that contain many misincorporated nucleotides (85). The priming and synthesis reactions for SV40 DNA replication in vitro begin after a time delay of 8 to 10 minutes (35, 37, 38, 88-90). This period has been designated the presynthesis stage. This delay can be avoided by a prior incubation with T antigen, the single-stranded binding protein RP-A, and topoisomerase I or I1 (25,33,34).This period involves the steps of origin recognition and unwinding, followed by polymerase a binding and primase action, as shown in Fig. 1.

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111. Mechanisms of Leading- and Lagging-strand DNA Synthesis

A. The Monopolymerase System The minimum complement of cellular proteins that carry out extensive bidirectional replication of SV40 include T antigen, DNA polymerase alprimase, topoisomerase I, and human SSB (RP-A) (91). As discussed below, hybridization analysis showed that larger products resulted from leading-strand synthesis, whereas shorter products were derived from lagging-strand synthesis (92). This is reminiscent of the situation in prokaryotes, in which the same DNA polymerase carries out leading- and lagging-strand DNA replication, with the necessary asymmetry of function provided by auxiliary subunits. However, the nucleus of eukaryotic cells contains three DNA polymerases, ci (93, 94), 6 (95),and E (96), each of which is required for viability in yeast. Their presence suggests that there is a more complex replication mechanism in eukaryotes. Alternatives include a role for all three polymerases in the process of chromosomal replication, or participation of one or more of the polymerases in a repair process that is critical for viability.

B. Polymerase 6 and PCNA First indications that chromosomal DNA replication might require more than one DNA polymerase came from the study of DNA polymerase 6 (97, 98). This nuclear enzyme, after purification from calf thymus, was distinguished from other polymerases by its intrinsic 3'-to-5' exonuclease activity and subunits of 125 and 48 kDa (98, 99). The larger catalytic subunit (100102) and smaller subunit (Antero So, personal communication) have now been cloned from human and calf. In early stages of purification, polymerase 6 utilized the substrate oligo(dT).poly(dA)(97, 98). Further purification reduced this activity, because of the removal of an important auxiliary factor. This auxiliary factor was later purified based on the ability to stimulate polymerase 6 synthesis on the homopolymer substrate (I03). Proliferating cell nuclear antigen (PCNA) had been under investigation for some years prior to the discovery of the 6 auxiliary factor, as a protein that might be involved in the control of cell growth. It appears at higher levels in tumor cells compared to normal cells (104-109). Immunostaining analysis showed that its level rises with cell growth (110-113) and DNA synthesis (114-119). The polypeptide is about 36 kDa and has been cloned from rat and human (120, 121) cells. Two important logical connections followed: the realization that PCNA is the auxiliary subunit of DNA polymerase 6 (103,122,123),and that PCNA is

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required for replication of SV40 in vitro (124, 125). These connections led to the proposal that DNA polymerase 6 and PCNA elongate the leading strand in SV40, whereas DNA polymerase a participates in lagging-strand synthesis (124, 126-128).

C. The Single-stranded DNA Binding Protein RP-A (RP-A, SSB) Single-stranded DNA binding proteins assist in the strand separation reactions necessary for DNA metabolism in prokaryotes and eukaryotes (10). The mammalian single-stranded binding protein RP-A was identified as an essential protein for SV40 DNA replication in vitro (43, 91, 129). It is a heterotrimer with subunits of 70, 32, and 14 kDa (43,129,130).The cDNAs for the subunits have been isolated (131-133) and coexpressed in E . coli to form an active complex (134).The genes for all three RP-A polypeptides are essential in yeast (135, 136). The DNA binding ability resides in the 70-kDa subunit (124,130,132).The 32-kDa subunit becomes phosphorylated at the beginning of S phase (137) suggesting cell-cycle-dependent regulation of function. RP-A has high &nity for single-stranded DNA, but low &nity for double-stranded DNA and RNA (138).Human RP-A forms two distinct complexes with single-stranded DNA (139).One complex cooperatively binds 810 nucleotides and the other binds 30 nucleotides with a preference for pyrimidine-rich sequences; it displays low binding cooperativity (139, 140). The binding-site size of the single-stranded DNA binding protein isolated from Drosophila was about 22 nucleotides (141). RP-A increases the fidelity of DNA synthesis by polymerase a from 2- to 8-fold in vitro (142). RP-A also stimulates the activity of calf thymus DNA and HeLa cell helicase E (145)and cx helicases A, B, C , D (143),and E (144), (146). RP-A stimulates pol-dprimase in a species-specific manner whereas its stimulation effect on polymerase 6 can be substituted by other singlestrand DNA binding proteins (147). Polymerase E activity from both yeast and human cells is significantly increased in the presence of RP-A (148). Aside from its role in DNA replication, RP-A is also thought to act in DNA repair (149, 150)and recombination (135, 151).

D. RF-C, RP-A, and the Assembly of the Highly Processive Leading-stra nd Complex A key to the understanding of the leading-strand replication mechanism was the discovery of RF-C. RF-C was identified as a necessary factor for complete SV40 DNA replication (152). Replication reactions performed in the absence of RF-C or PCNA resulted in accumulation of short products. Hybridization analysis showed that these were primarily lagging-strand products. RF-C is a multisubunit factor with five components of 140-145,

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40, 38, 37, and 36 kDa (153,154)in human cells. The structural and functional properties of RF-C are highly conserved from yeast to humans (155157). The genes for all the known subunits of RF-C have been cloned and sequenced from both human (153, 158-160) and yeast cells (161,162, and Bruce Stillman, personal communication). RF-C is also a DNA-dependent ATPase (163),with greater affinity for primer-template structures than for single- or double-stranded DNA. The ATPase activity is stimulated by PCNA (163).Binding analysis showed that the presence of ATP increases the specific interaction of RF-C with primertemplate junctions (164).PCNA interacts with the RF-C-primer-template complex. Specificity of primer-template junction recognition by RF-C is heightened by the presence of RP-A (164). Although PCNA stimulates highly processive DNA synthesis on oligo(dT).poly(dA) (103),Tsurimoto and Stillman (127) found that the action of polymerase 6, PCNA, and RF-C is necessary to allow highly processive synthesis on primed circular M13 DNA, making products thousands of nucleotides in length. RP-A, RF-C, and PCNA cooperatively stimulate the activity of DNA polymerase 6 (147,127).[RF-C has also been designated A1 by Hurwitz and colleagues (165)l. Other work (166)suggests that assembly of the leading-strand replication complex is the result of an interaction between RF-C, RP-A, PCNA, and DNA polymerase 6. An important role of RP-A is to control the use of primed single-stranded DNA by DNA polymerases. As the concentration of RP-A was increased (166),it first stimulated and then inhibited D N A polymerase a.Over the same concentration range, it thoroughly inhibited DNA polymerase 6, in the presence or absence of PCNA, RF-C, or both. However, when ATP was present with all four proteins, active polymerization occurred. When RF-C, RP-A, and PCNA were present, DNA polymerase a was still active for synthesis. However, addition of ATP stopped the reaction by polymerase a.The authors (166)concluded that RF-C and PCNA can form a primer recognition complex in the presence of ATP that allows primer binding and extension by polymerase 6 but not a. Polymerase 6 appeared to carry along the RF-C during synthesis, because addition of ATPyS immediately inhibited primer elongation.

E. Reconstitution of SV40 DNA Replication with Two DNA Polymerases The specificity for primer use conferred by RP-A and the characterization of the primer recognition complex prompted Tsurimoto and Stillman to propose the currently accepted mechanism by which polymerases a and 6 participate in SV40 replication (166).They examined replication of an SV40

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origin-containing plasmid in the presence of T antigen, topoisomerases I and 11, and D N A polymerase a. DNA synthesis required RP-A, and produced long leading-strand products and short lagging-strand products, as expected for the monopolymerase system. However, when the RP-A concentration was raised from the stimulation peak at 12.5 pg/ml to 50 pg/ml, the long products disappeared. Evidently high RP-A concentration blocked rebinding of polymerase a to the nascent leading strands. Addition of PCNA and RF-C eliminated long products, even at low RP-A levels. Most importantly, the addition of polymerase 6, in the presence of PCNA and RF-C, restored the synthesis of long products, even in the presence of high RP-A concentrations. DNA polymerase a was still necessary to synthesize significant amounts of short lagging-strand products. These observations led Tsurimoto and Stillman to propose the polymerase switching model (166) in which the leading strand is initiated by DNA polymerase a on an RP-A-coated template. After completion of the first Okazaki fragment, RF-C, driven by ATP, forms a complex with the primer terminus and then recruits PCNA to make the primer recognition complex. Polymerase 6 then replaces polymerase a,and begins highly processive synthesis in a complex with PCNA and RF-C. This model is depicted in Fig. 2. At the time of this work, polymerase a was proposed to carry out both priming and elongation of the lagging-strand segments. RF-C was thought to function as a stimulator of polymerase a (164, 166). More recent evidence based on investigation of completion of lagging-strand synthesis, discussed below, suggests that polymerase switching also occurs on the lagging strand.

F. Details of the Leading-strand Replication Process

Tsurimoto and Stillman (163) pointed out the remarkable functional resemblance between the interaction of DNA polymerase 6, PCNA, and RFC, and the mechanisms of bacteriophage T4 and E. coli DNA replication. The T4 gene 44/62 protein complex has ATPase and primer binding activities similar to those of RF-C (167-170). The T4 gene-45 protein is a functional and partial sequence homolog of PCNA that stimulates the ATPase of the 44/62 protein just as PCNA stimulates the ATPase of RF-C (167). Both proteins are needed to assemble a processive replication complex with the gene-43 DNA polymerase (167). In E. coli, the bulk of chromosomal DNA replication is carried out by DNA polymerase 111 (171).Components of this enzyme include an interactive group of the protein subunits y, 6, a', x and called the y complex (172).The y complex carries out an ATP-dependent assembly of the p subunit of DNA polymerase I11 onto primed DNA (173).

+,

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FIG.2. The polymerase switching mechanism

The p subunit is assembled into a toroidal hoinodimer (174) that can slide freely on DNA but cannot readily dissociate. It then interacts with the polymerizing subunit to make it highly processive for DNA synthesis. It has been proposed that PCNA is a toroidal homotrimer (174, 175) that can slide onto the ends of linear DNA, but must be assembled around closed circular DNA by the ATP-driven action of RF-C (176). Recent crystallization of yeast PCNA (1 77) verified the toroidal structure. Binding of polymerase 6 and possibly other replication proteins to PCNA locks them onto the template strand for highly processive DNA synthesis. Burgers and Yoder (176) postulated that PCNA could assemble into a highly processive complex with polymerase 6 and a linear homopolymer substrate because the PCNA could

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slide over the end of the linear template. Because primed M13 is circular,

the RF-C-mediated, ATP-dependent assembly of the PCNA toroid around the template is required.

G. DNA Polymerase E DNA polymerase E was first isolated from rabbit bone marrow as a polymerase containing an intrinsic 3’-to-5’ exonuclease (178). It has also been isolated from human placenta (179, 180), cultured cells (181, 182), calf (99, 183-189, and yeast cells (reviewed in 186). The major active-site subunits range from 140 kDa, for the major form in calf, to 258 kDa in humans. However, the calf has a minor form at 220 kDa, suggesting that the smaller form is a product of proteolysis (187). The yeast enzyme also appears as two forms, a tetramer with subunits of 200, 80, 30, and 29 kDa and a smaller form of 145 kDa (186).The 258-kDa catalytic subunit of the human enzyme has been cloned and sequenced (188). Unlike polymerase 6, the capacity of polymerase E to synthesize on oligo(dT).poly(dA) was retained throughout purification (189, 190). This suggests that polymerase E is active in the absence of PCNA. Indeed, it is also processive for 500-1500 nucleotides on primed single-stranded DNA (182, 191, 192), with little effect of added PCNA (185). Furthermore, addition of RF-C, ATP, and PCNA in various combinations to the yeast polymerase E did not significantly alter processivity, but did reduce salt sensitivity of the activity (148). In general, analyses of both human and yeast polymerase E (148) showed that approximately 200 mM salt inhibits synthesis on primed poly(dA) or M13 DNA. However, addition of SSB from one of several organisms (human, yeast, E . coli, or T4), with HeLa PCNA, A1 protein (RF-C), and ATP, allowed synthesis to occur. These components also stabilized binding of either polymerase 6 or E to the primer-template, although binding was still rather weak for polymerase E. These results suggest that DNA polymerase E and polymerase 6 may participate in similar reactions such as polymerase switching. Yeast polymerase E is encoded by a gene distinct from that of other DNA polymerases, and its disruption is lethal (96). Morrison et al. (96)also pointed out that defects in polymerase a,6 , or E all result in the same terminal cell morphology. This was a dumbbell-shaped cell with the nucleus at the isthmus. It is indicative of a defect in DNA replication (193). There is also evidence of involvement of polymerase E in repair (181). However, participating in repair does not detract from the likelihood of an involvement in replication, because polymerase OL and 6 are also implicated in repair (194196). Interestingly, PCNA is also required for DNA excision repair (196). This further suggests that either or both DNA polymerases 6 and E have dual roles in replication and excision repair. Mutations in many known DNA

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repair genes in yeast almost always result in viable cells (197), suggesting that cells can survive disruption of a polymerase solely involved in DNA repair. Of course, because repair processes are redundant, if polymerase E were required in all repair pathways, its absence could be lethal. DNA polymerase E was proposed to serve as the mammalian leadingstrand polymerase based on its requirement for viability and capacity for highly processive DNA synthesis (96). Meanwhile, Lee et al. (148) substituted DNA polymerase E for DNA polymerase 6 in the dipolymerase SV40 DNA replication system in oitro. Long products characteristically made by polymerase 6 disappeared. Overall, products were only about one or two times longer than those made by pol a alone, suggesting that polymerase E is not designed to carry out leading-strand synthesis. The somewhat longer products made in the presence of polymerase E were also dependent on the presence of polymerase alprimase. This result suggests that polymerase E had extended Okazaki fragments made by polymerase a. The likelihood that polymerases 6 or E complete Okazaki fragment synthesis is consistent with other observations (198)that synthesis of the longer lagging-strand fragments of SV40 replication in oitro is less sensitive to inhibition by butylphenyl-dGTP than that of the shorter fragments. Because this inhibitor affects polymerase a at considerably lower concentration than polymerase 6 or E, the result suggests that in their system polymerase 6 or E was adding to primers initiated by polymerase a. Furthermore, Bullock et al. (44) showed that antibodies to PCNA shorten lagging-strand SV40 segments made in oitro. This suggests that a PCNA-dependent enzyme was completing synthesis of these fragments. However, that polymerase also could be DNA polymerase 6. This was again pointed out by Waga et al. (199, 200), who failed to identify polymerase E as a protein that detectably improves the efficiency of production of form I closed-circular SV40 DNA in oitro. If polymerases 6 or E can be used interchangeably for completion of lagging-strand synthesis of SV40 DNA in oitro, the need for inclusion of polymerase 6 will make it difficult to verify the possible roles for polymerase &.

IV. Completion of Lagging-strand Synthesis

A. The Prokaryotic Mechanism

In E. coli, the lagging strands are elongated by DNA polymerase 111 up to the initiator RNA on the next nascent DNA strand. The initiator RNA is thought to be removed by the action of the 5’-to-3’ exonuclease of DNA polymerase I(10). The E. coli RNase H may participate in the process (201), but genetic experiments show that it is not essential (202). The 5’-to-3’

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exonucleases of both DNA polymerase I and the Taq polymerase are greatly stimulated by synthesis from the upstream primer to generate a nick (203). The coordinated action of synthesis and nuclease functions, termed nick translation (204), is thought to be the means of RNA removal. It also generates a nicked structure between the resulting two nascent DNA segments. DNA ligase then joins the DNAs. Polymerase I and the Taq polymerase also have an endonuclease function (203). A very specific substrate, consisting of two primers annealed to a template, is required. The downstream primer must have an unannealed 5’ end region. The upstream primer must be annealed with its 3’ end directly adjacent to the first annealed nucleotide of the downstream primer. The endonuclease then removes the unannealed region as an intact segment. The role, if any, of the endonuclease function in lagging-strand DNA synthesis is not known.

B. Reconstitution of the Joining of Mammalian Lagging-strand Products Requires a 5’-to-3‘ Exonuclease

There is no homolog of DNA polymerase I in mammalian cells. However, a thorough characterization of a mammalian 5’-to-3’ exonuclease suggests that it performs the same role as the 5’-to-3’ exonuclease of DNA polymerase I. A key observation was that a 5’-to-3’ exonuclease is required for generation of closed circular products during SV40 DNA replication in uitro (92). The authors began with the monopolymerase SV40 DNA replication system described by Wobbe et al. (91)utilizing SV40 origin-containing DNA, HeLa DNA polymerase dprimase, topoisomerase I, and the HeLa SSB (RP-A). This group of components produced form-I1 nicked double-stranded DNA products. Supplementation with HeLa DNA ligase, RNase H1, and topoisomerase I1 still did not lead to closure. However, HeLa extracts contained an activity that would allow generation of form-I DNA. Using closure as an assay a 5’-to-3’ exonuclease was purified (92) that w a s a monomeric protein of 44 kDa. It was a 5‘-to-3’ exonuclease that could degrade oligo(dA) or oligo(rA) annealed to poly(dT). Mononucleotide products were released from the 5’, but not the 3’ ends of the substrate. Primers not annealed to templates resisted cleavage. The preparation contained no phosphatase activity. RNase H1, isolated by a variation of the method of DeFrancesco and Lehman (205), stimulated priming and the subsequent DNA synthesis reaction severalfold. In the absence of the 5’-to-3’ exonuclease, products were made that spanned about half of the length of the SV40-origin-containing plasmid; a second population was about 200 nucleotides long (92).Hybridization experi-

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ments with leading- and lagging-strand probes verified that longer products were leading-strand intermediates, whereas the shorter ones were laggingstrand Okazaki fragments. Overall, these results implicated the 5’-to-3‘ exonuclease in the removal of initiator RNA. The 5’-to-3‘ exonuclease identified by complementation of SV40 DNA replication was identical to a cellular enzyme designated the “pL protein” (206).This protein aided the initiation of adenovirus DNA replication when the viral DNA lacked terminal protein. The nuclease acted by removing nucleotides from the 5‘ end of the double-stranded viral genome, providing a single-strand origin region. Working in parallel on DNA enzymes isolated from mouse cells, Goulian and Heard (207) came to virtually the same conclusions. They created a substrate to investigate the reactions of lagging-strand synthesis. DNA polymerase a/primase was used to randomly prime fd DNA and to continue synthesis until the DNA extended from each primer encountered the next adjacent downstream RNA primer. This substrate, when exposed to purified mouse DNA ligase I, RNase H1, and an extract fraction from mouse cells, was converted into closed circular DNA (207). This allowed an assay from which the essential factor in the extract could be purified. The factor was a 49-kDa 5’-to-3‘ exonuclease with virtually identical specificity for primed homopolymeric DNA as the HeLa exonuclease. They named it the cca (circle-closing activity) nuclease. A trade of the enzymes between the two laboratories showed that each could function in the other system. Experiments were then conducted to determine the respective roles of the 5’-to-3’ exonuclease and the RNase H1 in the process of RNA primer removal (208). In terms of nucleotide release, the exonuclease alone degraded about half of the RNA. The RNase H1 alone degraded 80 to 90% of the RNA. Together, these enzymes effectively removed all of the RNA. It was concluded that the 5’-to-3‘ exonuclease is relatively inert on the intact initiator RNA, with the observed activity likely caused by contaminating RNase H. They postulated that the RNase H removed most of the primer by endonuclease action. The remaining one or two nucleotides would then be susceptible to the 5’-to-3’ exonuclease. It is not clear why the original ribonucleotides in the initiator RNA resisted cleavage, whereas those remaining after RNase H action were susceptible. Harrington and Lieber (209) purified a mouse 5’-to-3’ exonuclease, designated FEN-1 (flap endonuclease), that appears to be the same as the cca nuclease. It did not degrade RNA oligomers annealed to DNA, which suggests that RNA length or sequence, or overall substrate structure, determines whether RNA is susceptible to cleavage. The genes for the mouse and human FEN-1 nucleases have recently been cloned, sequenced, and expressed (210).The murine FEN-1 gene is highly

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homologous with the S. cerevisiae genes Y K w l O and RAD2. RAD2 is essential for yeast excision repair (211) and was previously shown to be a nuclease (212). The expressed YKL510 peptide and a truncated form of RAD2 have structure-specific endonuclease activity (210). These results support a dual role for the 5'-to-3' exonuclease in D N A replication and excision repair.

C. Unique Substrate Specificity of the 5'-to-3' Exonuclease

An important feature of D N A polymerase I is its ability to carry out nick translation, the simultaneous synthesis from an upstream primer and degradation of a downstream primer. This results in movement of a nick in doublestranded D N A in the 5'-to-3' direction. This reaction is thought to be part of the process of initiator RNA removal and replacement of damaged DNA (10). The nick-translation phenomenon was investigated using the calf 5'-to-3' exonuclease, a homolog of the nucleases from HeLa and mouse cells. This 5'-to-3' exonuclease copurified through a number of chromatography steps with calf D N A polymerase E (213).A fraction containing the polymerase and the nuclease degraded a synthetic DNA or RNA oligonucleotide annealed to M13 DNA, but its activity depended on DNA synthesis from an upstream primer. Purification revealed the nuclease to be a monomeric protein that degraded a downstream deoxyoligomer irrespective of whether the DNA polymerase used to extend the upstream nucleotide was calf polymerase OL, 6 or E, E . coli polymerase I Klenow fragment, or T7 polymerase. If only one, two or three of the four deoxynucleoside triphosphates were used in the reaction, limiting the furthest position of extension of the upstream primer, the degradation of the downstream primer was likewise limited. These results suggested that the 5'-to-3' exonuclease was designed to work with a D N A polymerase for simultaneous synthesis and degradation. It was not clear from these results, however, what was responsible for the activation of the nuclease. One likely possibility is that the presence of a polymerase abutting the downstream primer is significant. Another is that the upstream primer had to be extended directly up to the downstream primer. We later found that the nuclease alone acts efficiently in a two-primer system if only a nick separates the primers (214). Under these circumstances the first nucleotide is rapidly removed from the downstream primer. However, there is essentially no further cleavage. Similarly, when a substrate with a one nucleotide gap between the primers, or one with no upstream primer at all, was tested, the activity of the nuclease was negligible. Parallel work (209) reached the same conclusions concerning the exonuclease from the mouse.

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These results explain convincingly why polymerization from an upstream primer stimulates exonuclease activity on a downstream primer. Polymerization continuously generates the nicked structure necessary for exonuclease action. It is also clear why either calf or prokaryotic DNA polymerases stimulated the calf exonuclease. As long as the polymerase could generate a nick, the nuclease would be stimulated. These observations show that the nuclease can act with any polymerase to carry out nick translation. The actual process of nick-translation was demonstrated using a substrate consisting of a synthetic template with a foldback upstream primer (215). A downstream primer separated by a four-nucleotide gap was then annealed. Either of DNA polymerases a,6, or E could extend the upstream primer to generate a nick that could be sealed by calf DNA ligase I. Podust and Hiibscher (216) obtained similar results, but had more difficulty obtaining ligation in reactions containing polymerase a, possibly because the polymerase is inefficient at complete gap filling (217). In fact, they point out that secondary structure in their template may have exacerbated this problem. These results show that the 5‘-to-3’ exonuclease does not work uniquely with only one DNA polymerase to carry out nick translation. Therefore, specificity of the nuclease cannot be used to define the DNA polymerase that participates in the joining of Okazaki fragments. Synthesis and ligation also occur in the presence of the calf 5’-to-3’ exonuclease (215).In this case, it appears that nucleotides are removed from the downstream primer prior to ligation. This means that synthesis from the upstream primer replaces the removed nucleotides, continually generating a nick that can be sealed. Because the nuclease works best on a nicked structure, genuine nick translation must occur, moving the nick into the downstream primer, followed by ligation.

D. Lagging-strand Completion in the Twopolymerase Reconstitution of DNA Replication in SV40 The most complete reconstitution of mammalian DNA replication to date was recently carried out in the SV40 system (199, 200). As with earlier reconstitution reactions, the components necessary for initiation at the origin and production of leading- and lagging-strand products were present. These included T antigen and PCNA, DNA polymerases a and 6, RF-C, PCNA, RP-A, and topoisomerases I and 11. However, the goal w a s to identlfy the additional components necessary to produce closed-circular-form-I DNA. Two fractions from human cells were identified that allowed generation of the form-I product. From one was purified a component named maturation factor I. This protein was a 44-kDa 5’-to-3’ exonuclease specific for double-

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stranded DNA. It was clearly identified by the authors as the same 5'-to-3' exonuclease described above. The other fraction contained DNA ligase I. Purified calf DNA ligase I was an effective substitute for this latter factor. Another important observation w a s that DNA ligase I rather than I11 was uniquely required for Okazaki fragment maturation during SV40 replication in uitro. Mammalian cells contain at least three DNA ligases (218, 219). Evidence suggests that DNA ligase I functions in DNA replication (220-223) and that DNA ligase I1 participates in repair (224).The role of ligase I11 is not known. The system of Waga et al. (199, 200) differs from that of Ishimi et al. (92) in that DNA polymerase 6, PCNA, and RF-C are present. Topoisomerase I included in the two-polymerase system allowed formation of the form-I product, whereas it inhibited its formation in the monopolymerase system. The latter system also needed a relatively high concentration of polymerase a and a low concentration of RP-A for polymerase a to carry out leadingstrand DNA synthesis. It was suggested (199) that the presence of DNA polymerase 6, PCNA, RF-C, and high levels of RP-A alter the reactions associated with initiator RNA removal and joining of Okazaki fragments. This seems particularly likely in the case of RP-A, which can bind directly adjacent to the initiator RNA. It was also pointed out that the preparation of DNA ligase I used contained RNase H1, and that the RNase H1 could have participated in the removal of initiator RNA (199, 200).

E. Polymerase Switching on the Lagging Strand Is Required for Ligation

Waga and Stillman (200) provided additional compelling evidence that a polymerase switching process is an essential feature of lagging strand synthesis. They used a synthetic Okazaki fragment model substrate consisting of a 445-nucleotide DNA template with a 30-nucleotide primer annealed to its 3' end. A 15-nucleotide RNA was annealed with its 3' end 227 nucleotides from the 5' end of the template, and then fully extended with DNA. Addition of RP-A, RF-C, PCNA, DNA polymerase 6, maturation factor 1, and a mixture of DNA ligase I and RNase H1 resulted in elongation of the upstream primer, RNA removal, and ligation. The same reaction could also be completed efficiently if DNA polymerase a was used in place of RP-A, RF-C, PCNA, and polymerase 6. However, addition of RP-A to the latter reaction partially inhibited product formation, and addition of PCNA and RF-C completely prevented generation of the ligated product. Therefore, the components of the reaction that load DNA polymerase 6 onto the primer also suppress the action of polymerase a.

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F. Specificities of RNase H1 and the 5’-to-3’ Exonuclease Suggest Their Functions in Okazaki Fragment Processing We used a synthetic Okazaki fragment substrate to model the initiator

RNA removal and ligation reactions (225). It consisted of a 13-nucleotide RNA transcript annealed to a longer DNA template, then extended by polymerization for 60 nucleotides. A DNA primer was annealed upstream on the same template, separated by 29 nucleotides from the 5’ end of the initiator RNA of the downstream primer. The complete reaction required removal of the initiator RNA, extension of the upstream primer, and ligation. Use of this substrate showed that some of our preparations of calf5’-to-3’ exonuclease contained a contaminating RNase H. Preparations with both activities removed the initiator RNA, whereas those with the exonuclease alone did not. Further purification resolved the two enzymes and allowed identification of the second activity as RNase H1 (226). In a time- and concentration-dependent manner, purified calf thymus RNase H1 degraded the RNA, generating a distinct degradation product. This product consists of the DNA portion of the primer with a single remaining ribonucleotide at the 5’ end. The rest of the RNA primer was released as an intact oligonucleotide, sustaining no further cleavage. The double-strandspecific 5’-to-3‘ exonuclease, added as a purified enzyme, removed the remaining monoribonucleotide. We used DNA polymerase E to generate the nick, which was then sealed by DNA ligase I. The exonuclease could potentially be stimulated by polymerization from the upstream primer, or by the cleaved RNA segment still annealed after the action of the RNase H. The polymerization steps could also be carried out by polymerases (Y or 6 (215). The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in uiuo. This series of reactions is depicted in Fig. 3. Eukaryotes contain two classes of RNase H, designated types 1 and 2 (227).RNase H 1 activity correlates with DNA synthesis, suggesting a role in DNA replication (227). DeFrancesco and Lehman (205) showed that RNase H from D.melanogaster not only removes initiator primers from Okazaki fragments in uitro, but also stimulates DNA synthesis by purified DNA polymerase dprimase. We have also examined mammalian RNase H1 cleavage products from three Okazaki fragment model substrates with different structures (228). Results show that the initiator RNA was removed in each case by a single cut made between the last two ribonucleotides upstream of the RNA-DNA junction (Fig. 3). The initiator RNA was released intact. Specific cleavage

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FIG. 3. Completion of lagging-strand DNA synthesis.

occurred even though the RNA segments ranged from 13 to 31 nucleotides in length. Furthermore, the position of cleavage was not influenced by the nucleotide sequence in the junction region. Cleavage specificity was lost if the RNA was not extended with DNA, or if there was a nick at the RNADNA junction. Specific cleavage was observed in the presence of Mg2+, or both Mgz+ and Mn2+. Cleavage with only Mn2+ was more random. Recent analysis of the solution structure of an Okazaki fragment model substrate indicates that the RNA-DNA junction has unique groove and bending features thpt could contribute to the specificity of RNase H cleavage

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(229). Comparison with E. coli RNase H or the human immunodeficiency virus reverse transcriptase RNase H showed that neither enzyme cut with the same specificity as the mammalian RNase H1 (228). HIV-RT made a preferred cut a fixed distance from the 5’ end of the RNA, whereas cleavages by E. coli RNase H appeared to occur at random positions. Overall, these results suggest that calf RNase H1 is designed to recognize the distinct structure of Okazaki fragments.

G. Endonuclease Function of the 5’-to-3‘ Exonuclease Another distinctive feature of the mouse 5‘-to-3’ exonuclease reported

by Goulian et al. (208) was its action on poly(dA-dT), a perfectly alternating double-stranded polymer. Surprisingly, the products were mostly the dimers d(T-A) or d(A-T), plus some alternating oligomers. This suggested that the enzyme has endonucleolytic activity. We later demonstrated a DNA endonuclease function in the calf 5‘-t0-3’ exonuclease that requires a substrate with a very specific structure (214). Cleavage requires a primer annealed to a template such that there is an unannealed region at its 5’ end. Furthermore, there must be a second primer annealed such that its 3‘ nucleotide is directly upstream of the first annealed nucleotide of the downstream primer. An endonucleolytic cleavage can then occur either just before or just after the first annealed nucleotide of the downstream primer. Endonuclease action removes unannealed segments of 2 to at least 12 nucleotides in length. It is likely that the unique structure of the alternating poly(dA-dT) allowed foldbacks that could transiently create the needed upstream primer and unannealed tail for the observed endonuclease action (208). Parallel work by Harrington and Lieber (209) showed that FEN-1 is also an endonuclease. It efficiently removes the unannealed segment of two primer substrates as described above, and does not cleave 3’ unannealed regions, Holliday junctions, or RNA 5’ unannealed regions (209). These observations suggest that, regardless of source, the mammalian 5‘-to-3’ exonuclease has endonuclease function. These specificities of nuclease action are not only unique compared to other mammalian nucleases, but are the same as the specificities of the 5’-to-3’ exonucleases of E. coli DNA polymerase I and Taq polymerase (203). This means that it is most likely that the mammalian 5’-to-3’ exonuclease is the functional homolog of the nuclease in the bacterial polymerase I. Because more than one of the mammalian DNA polymerases may perform nick translation, it is probable that the 5’-to-3’ exonuclease can serve all of them as a partner in that role. Rather than being physically attached to a single polymerase, the nuclease has evolved a specificity that requires coordination

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with any DNA polymerase in order to carry out cleavage associated with nick translation.

H. DNA Polymerase p Nuclei of eukaryotic cells also contain DNA polymerase p. It is smaller than the other nuclear polymerases, lacks associated nuclease activity, and does not undergo major changes in activity with cell growth (230). Rat and human forms have been cloned and expressed in bacteria (231), and the three-dimensional structure of the rat enzyme has been determined (232). Synthesis on single-stranded substrates and long gaps is distributive (231, 233-240). Synthesis on short gaps is most efficient and goes to completion (241).Interestingly, filling of short gaps of up to six nucleotides is processive, if the gap has a 5' phosphate (242). Photochemical cross-linking analysis indicates that on long gaps, the polymerase binds the 5' side of the gap. When the gap is shorter than six nucleotides, the 3' terminus can contact the polymerase, resulting in processive synthesis to fill the gap. These results suggest a role for DNA polymerase p in DNA repair rather than in replication. They are consistent with the observation that mutations in the polymerase p gene in yeast are not lethal (243). Also, recent experiments in which the level of polymerase p is down-regulated in cultured cells by antisense techniques show that the cells become sensitized to a wide variety of DNA-damaging reagents (Samuel Wilson, personal communication). Polymerase p might also participate in some aspect of gap filling associated with DNA replication, but in an optional pathway.

V. Regulation of Replication Reactions A. Specificity of Interaction of DNA Replication Components Analysis of the interaction specificity of a protein with other replication proteins can be used to verdy its unique participation at a particular step in DNA replication, compared to proteins with the same catalytic activity. One example is the interaction of initiation proteins with DNA polymerase a. Initiation at the SV40 replication origin requires a DNA polymerase OL from a cell type that is permissive for SV40 replication (244,245).This is not true of the bovine papilloma system, in which the E l protein, which performs the same functions as the SV40 T antigen, can initiate replication using polymerase OL from a nonpermissive cell type (246). Similarly the RP-A horn S. cerevisiae cannot substitute for the human RP-A in SV40 replication (247). Although the yeast RP-A supports T-antigen-directed origin unwinding, it

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does not allow normal T-antigen stimulation of priming by polymerase a. Additionally, T4 DNA replication proteins can substitute for DNA polymerase 6 and its auxiliary factors for leading-strand synthesis in SV40 DNA replication, but these proteins cannot support generation of form-I SV40, suggesting that they cannot interact with the components that remove initiator RNA and allow ligation (200).Also, DNA ligase I tlersus I11 was needed for the latter reaction. Evidently, depending on the system, there is a specificity of interaction that may be necessary, beyond the need for a particular catalytic action. Such observations prompted Waga and Stillman (200)to propose that the components of DNA replication are part of a single complex. This is similar to the proposed structure of prokaryotic replication forks ( I 73, 247-249). The model first proposed by Sinha et al. (247)for T4 DNA replication argues that the lagging strand is threaded through the complex in a loop and periodically released. A similar structure has been proposed for the bacterial and now the mammalian replication fork.

B. Regulation Mechanisms Only now are direct connections emerging between the growth regulation systems in eukaryotic cells and the protein components of the DNA replication machinery. Examples include phosphorylation of SV40 T antigen (as reviewed in 1I), phosphorylation of DNA polymerase a (250) and RP-A (138, and direct inhibition of PCNA by p21 (251). Phosphorylation plays an important role in the regulation of T antigen transcription and replication activities. Biochemical and mutational studies indicate that the direct effect of phosphorylation ofT antigen is on its binding ability to site I1 in the replication core origin (252).In contrast, the helicase activity and single-strand DNA binding activity of the T antigen were not affected (253-256). Phosphorylation of threonine-124 by the cell-cycleregulated cdc2/cyclin kinase was found to activate T antigen to initiate SV40 DNA replication (257). However, T-antigen kinase (casein kinase I) inhibits SV40 replication by phosphorylation of intact T antigen on serines-120 and - 123 (258).The cellular protein phosphotase designated RP-C, the catalytic subunit of protein phosphatase 2A, stimulates the initiation activity of T antigen (259, 260) and also can reverse the inhibitory effects produced by casein kinase I (261). DNA polymerase a is a phosphoprotein (250).Phosphorylation correlates with maximum synthetic activity (262,263).The level of the phosphorylated protein increases as cells move from quiescence to proliferation (264). The 180-kDa subunit is phosphorylated throughout the cell cycle, but is hyperphosphorylated in G,/M phase, whereas the 70-kDa subunit is phosphory-

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lated only in G,/M phase (250).The results suggest that the p34cAZ kinase is responsible for cell-cycle-related phosphorylation of DNA polymerase a (250). RP-A is also phosphorylated in a cell-cycle phase-specific manner (137), the 34-kDa subunit much more in S and G, phases than in G, phase (137). Both the 34- and 70-kDa subunits can be phosphorylated in vitro by the cdc2-cyclin B kinase complex (265, 266). G, extracts from HeLa cells, incubated in advance with human cyclin A, hyperphosphorylate the 34-kDa subunit (267). Phosphorylation was carried out by the cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). The tumor suppressor p53 protein regulates expression of the cyclindependent protein-kinase regular p21, a protein that inhibits SV40 DNA replication in uitro (251).Trimeric PCNA forms a one-to-one complex with p21, preventing stimulation of processive DNA synthesis by DNA polymerase 6 (251).This suggests a means by which p53 can regulate entry into S phase and damage control. Genetic analyses of the requirements for initiation of DNA replication offer the promise of elucidating the control of DNA replication. The power of this approach is exemplified in the analysis of origin-associated proteins. The genes ORC2 and ORC6 that encode ORC proteins are necessary for cellcycle progression and to bind the ARS A element in viuo (268-271). The minichromosome maintenance genes MCMI, MCM2, and MCM5 in yeast (272, 273) are essential for growth, and encode structurally related proteins necessary to maintain ARS-specific plasmids. The proteins localize to the nucleus between M phase and the beginning of S phase, binding tightly to DNA. They disappear from the nucleus at the onset of DNA replication, suggesting that they control the timing of replication initiation. These are characteristics of the “licensing factor” proposed to allow one round of chromosomal replication per cell cycle (274). Elucidation of the biochemical steps in the control of initiation of DNA replication will be the next major advance in this field. It offers the promise of more sophisticated efforts to prevent and treat diseases resulting from breakdown of growth regulation.

ACKNOWLEDGMENTS We thank John Turchi and Lynn Rust for critical reading of the manuscript, and Richard Murante for expert computer graphic illustrations. We are grateful to Bruce Stillman and Mark Kenny for reviewing our presentation. Also, our thanks to Shlomo Eisenberg for providing an excellent summary of the yeast origin literature. Support for this work was provided by National Institutes of Health Grant GM 24441.

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