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DNA arrested mutants of gene 59 of bacteriophage T4
VIROLOGY
69, 108-122 (1974)
DNA
Arrested
Mutants
of Gene
II. Replicative REEN
WU’
AND
59 of Bacteriophage
T4
Intermediates YUN-CHI
YEH
Department of Biochemistry, University of Arkansas School of Medicine, Little Rock, Arkansas 7%?Oi Accepted January 7, 1974 The abnormal DNA replication of bacteriophage T4 containing amber nonsense mutants in gene 69 (amC5, amHL628) has been studied. Zonal centrifugal analysis of the mutant intracellular DNA molecules isolated from a nonpermissive host, Escherichia coli B, reveals the following: (1) Parental mutant DNA as well as nascent mutant DNA can be recovered by sucrose gradient centrifugation as a fast sedimenting fraction (>1500 S) which is presumably a cell-membrane DNA replication complex. (2) At 2-3 min after initiation of viral DNA synthesis, a gradual accumulation of mutant DNA with a sedimentation coefficient of 63 S is observed. The molecular weight of this DNA is close to, or a little less than, that of a mature viral DNA, suggesting that less than one round of replication occurs before the complete arrest of DNA synthesis. (3) In contrast to what is found in wild-type infected cells, little or no long singlestranded DNA (concatemer) appears in mutant infected cells. Concatenated molecules of DNA, which have been proposed as intermediates in viral replication, are normally found in wild-type infected cells 7 min after infection. (4) Unlike the case of cells infected with mutants of gene 46 and 47, addition of chloramphenicol prevents premature release of the mutant DNA of gene 59 from the fast-sedimenting fraction (>1500 S). INTRODUCTION
Studies of phage T4 DNA synthesis in Escherichia coli have shown that in addition to replication mechanisms, recombination and repair are also involved (Cohen, 1968; Mosig, 1970; DaVern, 1971). These mechanisms are evidenced by the various abnormal phenotypes found among many of the conditional lethal mutants isolated by Epstein et al. (1963) that affect DNA synthesis. A multifork mode of replication occurs in T4-infected cells (Werner, 1968). Recently a bidirectional replication mechanism has also been suggested by electron microscope studies of replicative intermediates (Delius et al., 1971). Physical studies of replicative intermediates (Frankel, 1968; Miller et al., 1 Present address: Department of Biology, University of California, San Diego, La Jolla, California 92037.
1970) revealed that fast sedimenting DNA molecules are associated with the cell membrane, as was observed in various systems including E. coli (Rosenberg and Cavalieri, 1968; Fuchs and Hanawalt, 1970; Stratling and Knippers, 1971), B. subtilis (Sueoka and Quinn, 1968; Yamaguchi et al., 1971), pTX174 (Sinsheimer et al., 1968; Salivar and Sinsheimer, 1969), and S13 (Shleser et al., 1969). Frankel (1968) and Miller et al. (1970) have observed concatemers, replicative intermediates which are present 7 min after infection. They have shown that the strands sediment rapidly because they are concatenates of two or more genomes, not because of an altered conformation of the T4 DNA molecules. We are interested in the mechanisms involved in DNA replication following initiation, and have studied abnormal DNA replicat,ion by mutants of gene 59 (amHL108
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
REPLICATIVE
INTERMEDIATES
628, umC5). The mutants map between the maturation defective (MD) mutant gene 33 and recombination defective mutant gene 32 (Edgar and Wood, 1966). We have reported that the arrested synthesis of DNA caused by mutations in gene 59 can be restored by addition of chloramphenicol or by mutations in gene 55 and 33 (Wu et al., 1972). It is suggested, therefore, that at least one late protein is required for the expression of the mutant gene 59 phenotype. The present work focuses on physicochemical studies of DNA synthesis by umber mutants of gene 59 (amC5, ccmHL628) in the nonpermissive host, E’. coli B. We shall describe the abnormal dissociation of mutant DNA from the membrane-replication complex and also defects in concatemer formation. A possible role of gene 59 in the continuation of DNA synthesis will be discussed. There has been a preliminary account of some of these experiments (Wu and Yeh, 1970). MATERIALS AND METHODS Bacteria and bacteriophage. E. coli B strain Tr201, a thymine-requiring mutant obtained from Dr. G. R. Greenberg, was used as a nonpermissive host for amber phage mutants; E. coli CR63 (Stir+) was the permissive host used for growing the amber mutants. Two T4D amber mutants in gene 59, amHL628 and umC5, were supplied by Dr. R. Edgar. We have purified the mutants genetically by backcrossing twice to wildtype T4D. Also T4 umB272, which is defective in gene 23 (major head protein), was used. A double mutant amC5-amB272 was constructed by genetic recombination. Phage lysates were prepared in liquid culture and purified by differential centrifugation (Adams, 1959). Chemical reagents. All radioisotopes were from the New England Nuclear Company and all commercial enzymes were from the Worthington Biochemical Corporation. Media. Vogel and Banner medium (1956) was used in the isotope labeling experiments. It was made by mixing 0.1 ml. of 20 % glucose, 0.1 ml of 5 % casein hydrolyzate and 10 ml of 1 X salt solution (50 X salt solution was prepared by dissolving 10 g of MgSOd.
OF T4 GENE 59 MUTANTS
109
7HzO. 100 g of citric acid *Hz0 and 500 g each of KZHPOJ and NaNH4HP04. 4Hz0 in 670 ml water). This mixture was named 1 X CT- medium. The medium containing thymine or thymidine (5 pg/ml) was named 1 X CT+. Growth and infection of cells. An overnight culture of E. coli was diluted 50 times with fresh 1 X CT+ medium. The bacteria were grown with vigorous aeration at 37” until the optical density reached 0.8 at 590 nm on a Zeiss spectrophotometer, corresponding to a viable count of about 5 X 108 cells/ml. The cells were centrifuged and resuspended in an equal volume of 1 X CT- medium in the presence of 20 pg/ml D,I,-tryptophan. Phage was added at a multiplicity of approximately 5 phage particles per bacterium. Three minutes after infection, 14C-thymine (1 &J/3 pg/ml) or 3H-methylthymine (10 &i/3 pg/ml) was added. In all experiments, the fraction of bacteria surviving was less than l%, and in most cases, it was considerably less. In labeling the DNA of the parental phage, the cultures of E. coli were infected at a multiplicity of 0.1 phage per bacterium in the presence of 3H-thymine (50 pCi/3 pg/ml) or 14C-thymine (5 &i/l5 pg/ml). After 5 hr of incubation, a few drops of chloroform were added and aeration was continued for an additional 10 min and then the lysate was purified (Adams, 1959). In most cases the labeled phage was used within a week. Treatment of radioactive samples. The radioactivity in acid-insoluble fractions was determined by pipetting 0.1 ml of a sample onto a 2.5 cm disk of Whatman No. 3 filter paper. The disks were washed for 10 min in three successive 209ml volumes of 5% trichloroacetic acid (TCA) followed by two successive rinses in 200 ml of 95 % alcohol. This is a slight modification of the procedure described by Bollum (1966). The filters were then dried and placed in vials containing 5 ml each of toluene phosphor (4g/l PPO and 50 mg/l POPOP) and counted in a Nuclear Chicago liquid scintillation counter. Sucrose gradients and centrifugation. For neutral sucrose gradients, 5 ml linear 5 to 20 % sucrose gradients were layered over 1 ml saturated sucrose. Sucrose solutions were
110
WU ilND
prepared in 1 X SSC solution (0.15 N NaCl and 0.015 N sodium citrate, pH 7.5) in the presence of 0.015 n/r EDTA. For alkaline sucrose gradients, linear 5 to 20 % sucrose gradients were also layered over 1 ml of saturated sucrose in order to improve sample recovery. Alkaline sucrose solutions were prepared in 0.05 M NasP04 buffer (pH 12.2) in the presence of 0.015 M EDTA. Centrifugation was performed at 25,000 rpm for 30 min and 30,000 rpm for 60 min for neutral and alkaline gradients, respectively, in a SW 40 rotor of a Beckman Model L2-65B preparative ultracentrifuge. The tubes were pierced and 8 drops per fraction were collected onto Whatman No. 3 filter papers. After drying, the radioactivity of the acid-insoluble fractions was assayed. Lysing procedures. The procedure of Frankel (1966) was used for lysing cells before neutral sucrose gradient centrifugation. Cells were pipetted into an equal volume of a solution consisting of 0.1 M EDTA, 0.1 M NaCN, and 200 pg egg white lysozyme/ml at pH 8.5, and the mixture was placed in a 65” water bath. Partial clearing occurred after 1 min, at which time sarkosyl NL 97 was added to 0.1%. Incubation was continued for an additional 10 min and the resultant clear lysate was removed from the water bath and cooled to room temperature. In preparation for alkaline sucrose gradient centrifugation, we used two lysing procedures, one described by Frankel (1968) and another by Miller et al. (1970). In the former method the cells are treated with 1 mg lysozyme/ml, 3% sarkosyl NL 97 (Geigy Industrial Chemicals) and 0.2 N NaOH. In the latter method the cells are cooled in an ice bath and then treated with 100 pg lysozyme/ml for 10 min, after which Triton X-100 is added to a concentration of 1 %, and 10 min later the DNA is denatured by addition of NaOH to 0.4 N. In our hands, the lysozyme-Triton method caused less hydrodynamic shearing than the method described by Frankel. Isolation of replication complex. Two methods were used in this study. One was adapted from Miller and Kozinski (1970), and was similar to the lysozyme-TritonNaOH method, except that NaOH was not
YEH
added and the Triton X-100 treatment was prolonged to 4 hr. The other was a slight’ modification of Knippers and Sinsheimer’s method (1968). Infected cells (5 X 108 cells) were suspended in 0.25 ml. of 20% sucrose solution (0.05 &! Tris, 0.05 1M NaCl, 0.05 M EDTA, pH 8.0) ; 0.05 ml of 2 mg lysozyme/ ml was added, and the mixture was incubated for 20 min at 37”. Next., 0.2 ml of 5 % Brij 58 (Atlas Chemical Industries, Inc.) was added, and incubation continued for 10 more min. Finally, 0.5 ml of the same TrisNaCl-EDTA solution was added, followed by gentle mixing. This method will be designated lysozyme-Brij method. CaZcuZation. The data from sucrose gradient centrifugation were analyzed by the use of a computer program. Molecular weights and sedimentation coefficients were calculated according to t’he equation of Burgi and Hershey (1963). The number of nicks in the chromosome was estimated using the method of Litwin et al. (1969). The molecular weight of T4 standard DNA was taken as 118.3 X lo6 for double-stranded DNA (Gray and Hearst, 1968). The s value of T4 mature DNA is taken as 63 S (Frankel, 1966). RESULTS
Awested DNA Synthesis After the initiation of DNA synthesis by amber mutants of gene 59 (amC5, amHL628) in the nonpermissive host E. coli B, it is not known whether synthesis is completely stopped or a steady state of synthesis and degradation of DNA is maintained. The kinetics of DNA synthesis in E. coli B (thy-) infected with T4 am+ and T4 amC.5 (gene 59) were studied by pulse-labeling with 3H-methylthymine into acid-insoluble material. The results (Fig. 1) show that initially the mutant synthesizes DNA at the same increasing rate as the wild-type, but it reaches its maximum rate of incorporation at about 7-8 min after infection following which the rate decreases rapidly. In contrast, wild-type infected cells show a rate of synthesis that increases to a high plateau level. This result, and also the one obtained by continuous labeling (Fig. I), clearly indicate that the observed arrest of DNA synthesis is not due to a steady state caused
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TIME
AFTER
INFECTION
INTERMEDIATES
(MIN)
FIG. 1. Kinetics of DNA synthesis in cells infected with wild-type T4D and T4 amC5 (gene 59). Thymine-2-W (1 &i/3 pg/ml) was added at the beginning of infection. At various times, 0.2 ml of infected culture was pulse labeled for 30 set with ZH-thymine (20 &i/O.14 rg). The incorporation was stopped by adding an equal volume of cold 0.1 M KCN-tJ.05 EDTA pH 8.0. Samples of 0.1 ml were taken to measure radioactivity in the acidinsoluble fraction as described in Materials and Methods. (O), 3H-thymine; (0), thymine-2-W incorporated into T4 amC5 infected cells. (A), 3H-thymine; (a) thymine-2-14C incorporated into T4D infected cells.
by synthesis and degradation. It is seen that the mutation in gene 59 has an effect by 2-3 min after DNA synthesis is initiated and eventually causes a complete arrest of DNA synthesis. Furthermore, the newly synthesized mutant DNA is quite stable with no detectable degradation. Premature Release of Mutant Replication Complex
DNA
from
Replicative intermediates in Brij-58 treated lysates. The involvement of a DNA-protein complex has been demonstrated in T4 phage replicative cycles (Kozinski and Lin, 1965). The complex is thought to be attached to the cell membrane and is frequently referred to as a “membrane” complex (Frankel, 1968; Altman and Lerman, 1970; Earhart, 1970). Replicative intermediates of T4 amC5 in-
OF T4 GENE 59 MUTANTS
111
fected cells have been studied by using nonionic detergents (Brij 58 or Triton X-100) for lysing the infected cells and analyzing in neutral sucrose gradient sedimentation. When lysozyme-Brij 58 is used for lysing medium immediately after pulselabeling (Fig. 2a, f), most of newly labeled DNA (from 4.5 to 5 min after infection) in both T4 am+ and am- infected cells sediment faster than 1500 S and can be recovered from the cushion; the exact s value cannot be measured. Other investigators have found in T4-infected cells a similar complex with reports of sedimentation values up to ~800 S, even though the isolation procedures were somewhat different (Altman and Lerman, 1970; Earhart, 1970; Miller and Kozinski 1970; Shah and Berger, 1971). This fast-sedimenting property indicates that a complex of labeled DNA with proteins or membrane is formed. Otherwise, the sedimentation value of replicative DNA would be smaller. The premature release of the mutant DNA from the replication complex is evidenced by the following. About 24% of 3H-labeled DNA in T4 amC5 infected cells obtained after a 2.5-min chase (7.5 min after infection) is released from the complex (> 1500 S) and therefore remains close to the top of gradient (Fig. 2b). This slowly sedimenting material is found close to the top of gradient (Fig. 2b). This slowly sedimenting material is found close to the position of mature T4 DNA (63 S). The fraction of this prematurely released DNA (63 S) increases as infection continues. About 35 % and 40% of the labeled DNA are released from the complex (> 1500 S) 9.5 and 15 min after infection, respectively, in mutant-infected cells (Fig. 2c-d); then a drop to 34% occurs at 30 min (Fig. 2e). However, this premature release of DNA from the fast-sedimenting complex is not observed throughout the replicative cycle in wild-type infected cells (Fig. Zf-j). ’ In addition to this 63 S material, a second peak of DNA appears in mutant-infected cells 15 min after infection (Fig. 2d). It sediments as rapidly as 14C-labeled phage particles (-800 S) and resists DNase digestion. This property suggests that the 800 S
WU AND YEH
112 T4mC5
T4D
40 20 0 40 20 0 4BOTTOM
FRACTION
NUMBER
FIG. 2. Neutral sucrose gradient analysis of intracellular DNA lysed by lysozyme-Brij 58 method. Escherichia coli B Tr201 cells were infected with T4D or T4 czmC5. *H-Thymine (20 &i/ml) was added at 4.5-5.5 min after infection. The pulse was terminated by the addition of an equal volume of medium containing
thymine
(2 mg/ml).
Samples of infected
cells at various
times after infection
were lysed by the
lysozyme-Brij 58 method in the presence of ‘Glabeled T4 phage. Samples were centrifuged for 30 min at 10,OCOrpm as described under Materials and Methods. (a) to (e) were DNA extracted from cells infected with T4 a&5. (f) to (j) were DNA extracted from cells infected with T4D. (h) and (i) were independent experiments from the rest. The sedimentation is from right to left in this and subsequent figures. a---@, aH-labeled DNA; O---O, ‘“C-labeled reference T4 DNA. DNA is a mature phage particle, and indicates that maturation occurred even in the mutant phage. As shown in Fig. 2 (e and j) 41% and 71% of 3H-labeled DNA are encapsulated into phage particles after 30 min in mutantand wild-type infected cells, respectively. This observation is consistent with the burst size in E. coli B of this mutant phage of 10 to 15 infective centers/ bacterium.
This dissociation of the replicative complex formed by the newly synthesized DNA suggests that the product of gene 59 is required for stabilizing the replication complex. In order to investigate whether the premature release of T4 amC5 DNA from the membrane complex is due to the instability of the complex or due to encapsulation, an amber mutation in gene 23 (head protein)
REPLICATIVE
INTERMEDIATES
was introduced by genetic crossing. DNA encapsulation is inhibited in this double mutant (T4 amC5 -amB272, gene 59-23) ; however, the premature release phenomenon is still observed in mutant-phage-infected cells (Fig. 3). In addition, 90% of the amB272 DNA still remains in the fast sedimenting complex (2 1500 S). This result shows that the maturation process is not involved in the expression of premature DNA release. Furthermore, it is possible to eliminate the mature phage progeny from the replicative intermediates. Replicative intermediates in Triton X-100 T4
OF T4 GENE
59 MUTANTS
113
treated lysates. To obtain further information about replication complexes and premature DNA release, another nonionic detergent, Triton X-100, is used to isolate the replication complex from mutant-phageinfected cells. The experiment, similar to that of Fig. 2, is carried out in cells infected with the double mutant amC5-amB272. Immediately after a short isotope pulse (4.5-5.5 min after infection), most of the labeled replicative DNA sediments as rapidly as the complex (> 1500 S) isolated by the lysozyme-Brij 58 method (Fig. 4a). After a 2.5 min chase (7.5 min after infection), the
amC5-am8272 -amC5-018272
14 amB272 -
t
BOTTOM
FRACTION
NUMBER
FIG. 3. Neutral sucrose gradient sedimentation of intracellular DNA lysed by lysozyme Brij 58 method. Escherichia coli B Tr291 was infected with T4 amC5-amB272 (gene 59-23) or T4 amB272 (gene 23, used as a control). Conditions of isotope labeling and centrifugation were as described in Fig. 2. Cells were lysed by the lysozyme-Brij 58 method in the presence of ~KXabeled reference T4 DNA. In (a) to (d), DNA was extracted from T4 amC5-amB272 infected cells. In (e) and (f) DNA was extracted from 0, 8H-Labeled intracellular DNA; O-O, “C-labeled reference T4 T4 amB272 infected cells. l DNA.
114
WU AND YEH T4
a~C5-0~~1B272
w a
T4 amB27
f
2
w
BOTTOM
FRACTION
NUMBER
FIG. 4. Neutral sucrose gradient sedimentation of intracellular DNA lysed by the lysozyme-Triton X-100 method. Intracellular DNA was labeled (4.5-5.5 min after infection) and chased as described in Fig. 2. DNA was isolated by the lysozyme-Triton X-100 method in the presence of ‘%-labeled reference T4 DNA. In (a) to (c), DNA was extracted from T4 amC5amB272 infected cells. In (d), DNA was extracted from T4 amB272 infected cells. O--e, aH-labeled intracellular DNA: O-0, r*C-T4 reference DNA.
labeled 1500 S complex is dissociated into 850 S DNA and 63 S DNA (Fig. 4b). Though the 850 S material sediments close to the position of mature phage DNA particles, it is not the mature phage because of its sensitivity to DNase digestion. Finally, almost 88 % of the labeled DNA was released from the fast-sedimenting complex after 30 min of infection (Fig. 4~). In contrast, DNA from cells infected with umB272 phage sediments faster than 1500 S (Fig. 4d). These results are consistent with those obtained with the Brij 58 procedure as described in the preceding section. Both procedures gave the same gradual accumulation of 63 S DNA 2-3 min after the initiation of replication. Effect of chloramphenicol on premature release of mutant DNA. Previously, we
showed that chloramphenicol (CM) added 5-12 min after infection rapidly restored DNA synthesis, which is otherwise arrested after 12-14 min (Wu et al., 1972). The effect of CM on the premature release of mutant DNA was investigated. Chloramphenicol was added at set intervals to cells infected with the double mutant amC5-amB272. After 30 min the intracellular DNA was isolated by the lysozyme-Brij 58 procedure. Figure 5 shows the effect of chloramphenicol on the formation of the replication complex. It is seen that the addition of chloramphenico1 prior to 7.5 min after infection prevents premature release of mutant DNA from the replication complex (> 1500 S). However, when chloramphenicol is added 10 min after infection (Fig. 5c), 27 % of the replicative DNA is released. Since chloramphenicol is known to inhibit protein synthesis, it is sug-
REPLICATIVE
INTERMEDIATES
FRACTION
OF T4 GENE 59 MUTANTS
115
NUMBER
FIG. 5. Effect of chloramphenicol on the dissociation of the replication complex in T4 amC5 infected cells. Escherichia coli B Tr201 was infected with T4 amB272 or T4 amC5-amB272. Intracellular DNA was labeled at 4.5 to 5.5 min after infection as described in Fig. 2. Chloramphenicol (lOOpg/ml) was added at various times after infection and the cells were lysed 30 min after treatment. The sedimentation was carried out in a 5 to 200%neutral sucrose gradient. Arrows indicate the position of reference T4 DNA.
gested that at least one protein, synthesized beginning approximately 7.5 min after infection, participates in the dissociation of the replication complex in cells infected with gene 59 mutants. Furthermore, the restoration of DNA synthesis by chloramphenicol as well as formation of a stable replicative complex suggests that the fast sedimenting complex (> 1500 S) is essential for continuous viral DNA replication. Absence of Concatemer Formation To determine further the function of gene 59, we have studied the replicative intermediates of DNA molecules which are longer than one phage DNA equivalent unit. Two kinds of DNA molecules that are longer than one phage equivalent unit have been found in T4 phage-infected cells (Frankel, 1968; Murray and Mathews, 1969). One is formed within the first 5 min after infection, which is just before normal replication has commenced (Murray and Mathews, 1969). The other kind, originally described by Frankel (1968), is detectable 7 min after infection. The latter has been named “concatemer” and consists of several DNA molecules joined covalently end to end. Neutral sucrose gradient sedimentation. Figure 6 shows the sedimentation profile of mutant phage intracellular DNA in neutral
sucrose gradients. E. coli B Tr201 was infected with the double mutant amC5anB272. Phage DNA was labeled at the onset of DNA synthesis (from 3.5 min to 4.5 min infection at 37°C) with 3H-thymine, followed by a chase with cold thymine (1 mg/ml). Immediately after pulse-labeling, the newly synthesized DNA in mutantinfected cells sediments close to the reference T4 DNA (63 S) (Fig. 6a). It is very difficult to observe the difference in position between this intracellular DNA and the reference DNA, but occasionally there is a difference of one fraction in the gradient. This material corresponds to the long DNA described by Murray and Mathews (1969) that is 6 % longer than mature T4 DNA. To date, no mutant has been found defective in the synthesis of this long DNA (Murray and Mathews, 1969). At 6 min after infection (1.5 min chase), the labeled DNA sediments faster at a position that corresponds to 200 S material (Fig. 6b). This 200 S DNA, is the proposed precursor of mature phage DNA in wild-type T4-infected cells (Frankel, 1968). However, this fast sedimenting material (200 S) which appears in the early stage of the replicative cycle is transient in mutant infected cells. At 7.5 min after infection, 38 % of 3H-labeled DNA is released from 200 S DNA and has a sedi-
116
WU AND YEH T4amC5-amB272 al 4.5rnl”
---BUTTOM
T4amB272 t
FRACTION
b) 6 Omw
g)6.0msn
NUMBER
FIG. 6. Neutral sucrose gradient sedimentation of the mutant mtracellular DNA lysed by the lysoayme-sarkosyl-high temperature method. Escherichia coli B cells were infected with T4 amCbamB272 or amB272 as a control. *H-Thymine (25 &i/ml) was added at 3.5 min after infection and the incorporation was terminated by the addition of thymine (1 mg/ml) as described in Fig. 2. Intracellular DNA was isolated at various times after infection by the method described by Frankel (1966). Centrifugation was carried out in a SW 40 rotor at aspeed of B,ooO rpm ior 3Umin through & 5 to f20ya neutral swrrxse gradient. O-0, aH-labeled DNA; O-O, 14C-labeled reference T4 DNA.
mentation value similar to the marker T4 DNA (63 S, Fig. 6~). The fraction of 63 S DNA gradually increases until at 13 min after infection (Fig. &), most pulse labeled DNA appears near 63 S position, and this sedimentation profile does not change further (Fig. Sf). In contrast, DNA from cells infected with umB272 phage sediments at 200 S without any release of 63 S fraction during the replicative cycle (Fig. 6g-i). Although it appears that two kinds of replicative structures are generated in mutant-infected cells, one at 63 S and the other at 200 S, a pulse-chase experiment at
7.5 min indicates that the 63 S DNA is produced early in the replicative cycle as a dissociation product of the 200 S DNA (Fig. 7). This observation and the results shown in Fig, 6 suggest that one or more steps in the formation of 200 S DNA is defective in this mutant. Alkaline sucrose gradient sedimentation. Long strands of phage DNA have been observed in alkaline sucrose gradient (Frankel, 1968; MilIer et al., 1970). We have used the lysozyme-Sarkosyl-NaOH method to estimate the rate of joining of fragmented DNA and the size of replicative DNA in mutant-
REPLICATIVE
INTERMEDIATES 635
i
i.
.L IO
20
tBOTTOM
FRACTION
~ 30
40
NUMBER
FIG. 7. The fate of 290 S DNA in T4 amC5 infected cells. At 7 min after infection, mutant infected cells were pulse labeled with XI &i/ml for *H-thymine for 30 see (0), then chased with thymine (1 mg/ml) for 10 min (0). The intracellular DNA was isolated by lysozyme-salkosyl-high temperature method and sedimented through a 5 to 20y0 neutral sucrose gradient as described in Fig. 6. The arrow indicates the position of reference T4 DNA in a parallel tube.
and wild type-infected cells. Almost 50 % of the labeled DNA isolated immediately after pulse labeling (4 to 5.5 min) sediments in alkaline sucrose gradient slower than the position of 0.55 of reference T4 DNA (&‘D?) (Fig. Sa, e). This value corresponds to a molecular weight of about 13 million daltons (Abelson and Thomas, 1966). The fragmented DNA appears to be gradually joined. The second peak (Dz/D~ = 0.7) of DNA fragments is observed after 7.5 min infection in both types of phage-infected cells (Fig. 8b, f). This DNA of molecular weight 25 million is continuously joined in the wild type-infected cells until, at 12 min 2 DZ equals the distance from the top of gradient of the replicative DNA and DI equals the distance from top of the gradient of the standard phage DNA molecules.
OF T4 GENE 59 MUTANTS
117
after infection, most of the labeled DNA sediments faster or at the same rate as reference T4 DNA (Fig. Sh). In contrast to these results, the joining of fragmented DNA is slow in the mutant; after 12 min infection, 30 % of the labeled DNA was still in the position corresponding to Dp/Dl = 0.7 (Fig. Sd). In a separate experiment, the same result was obtained showing the joining of fragmented DNA (corresponding to 25 million daltons) is slow in mutant-infected cells. For comparison, wild-type phage DNA was continuously labeled by 2J4C-thymine and mutant DNA was labeled by 3H-thymine after infection. Samples from these two cultures were mixed and lysed by the lysozyme-Triton X-lOO-NaOH method. At 11 min and 15 min after infection, one-third of the replicative DNA in the mutant- and wild type-infected cells sedimented to a position corresponding to 25 million molecular weight (DJDI = 0.7, Fig. 9a, b). For wild-type phage this fragmented DNA is joined rapidly, only 16 % and 10% of this DNA being left after incubtation for 20 min and 30 min, respectively (Fig. 9c, d). In comparison, 22% of mutant DNA remains fragmented even 30 min after infection (Fig. 9d). This slow joining is consistent with the UV sensitivity found in the gene 59 mutants in which these mutants cannot repair damaged DNA caused by UV irradiation (Wu and Yeh, to be published). In addition to the defect in repairing fragmented DNA either after UV-irradiation or after normal infection with the mutant, there is an obvious difference in the formation of single-stranded DNA longer than one unit length of T4 DNA (the concatemer form). This form can be detected in wild-type infected cells 7.5 min after infection (Fig. Sg, h and 9) DNA twice as long as the mature phage DNA (DJDI = 1.29) is barely detected in mutant replicative cycles using either the lysozyme-SarkosylNaOH method (Fig. 8) or the lysozymeTriton X-160 method (Fig. 9). This observation corresponds well with the neutral sucrose condition in which DNA synthesized in this mutant has shorter strands during formation of the replicative cycle.
118
WU AND YEH
t
BOTTOM
FRACTION
NUMBER
FIG. 8. Alkaline sucrose gradient sedimentation of intracellular DNA lysed by the lysozyme-sarkosyl-NaOH Method. Intracellular DNA in phage-infected cells was labeled with 3H-thymine (2Q&X/ml, 4.5-5.5 min after infection) and chased with thymine (1 mg/ml). Sedimentation was carried out at a speed of 30,000 rpm for 60 min through a 5 to 20% alkaline sucrose gradient. In (a) to (d), DNA was extracted from cells infected with T4 amC5 at various times after infection. In (e) to (h), DNA was extracted from cells infected with T4D. The arrow indicates the position of 0.7 (02/01), except in (a) and (e), which show the position of 0.55 (Dt/Dl). +--0, 3H-labeled DNA; O-O, “C-labeled reference T4 DNA.
This result leads us to conclude that the gene 59 mutant is defective in the formation of long-chain concatemers. Integrity of Parental Mutant Phuge DNA The inability to detect single chains longer than the marker T4 DNA, as well as the slow repairing of fragmented DNA in the mutant, can be explained if, at early times after infection, discontinuities are introduced into the parental template molecules and replication occurs on partially degraded templates. Therefore, the integrity of parental DNA was investigated at early times after infection. It is clear that the parental DNA, after both mutant and wildtype infection, have similar sedimentation profiles, except in the formation of longstranded DNA, which is barely observable in the mutant-infected cells (Fig. 10). However, most of mutant parental DNA has the same sedimentation rate as reference T4 DNA. Therefore, it is concluded that in the absence of gene 59 function, early break-
down of the template molecules does not appear to account for its inability to synthesize long, single-stranded DNA and to repair fragmented DNA. DISCUSSION
Since its first proposal (Jacob et al., 1963), association of replicative DNA with a cell component, presumably the cell membrane, has been reported in many species including prokaryotic as well as eukaryotic cells (Goulian, 1971; Klein and Bonhoeffer, 1972). However, the nature and role of membrane components in DNA synthesis have not been elucidated. Using two nonionic detergents, Brij 58 and Triton X-100, we have shown that a replication complex (> 1500 S) is involved in phage T4 replication. Similar results have been reported (Altman and Lerman, 1970; Diggelmann et aE., 1970; Miller and Kozinski, 1970; Shah and Berger, 1971). Stratling and Knippers (1971) found that this complex (> 1500 S) isolated by the
REPLICATIVE
INTER.MEDIATES
OF T4 GENE 59 MUTANTS
119
but it is evident that they play an important role in T4 DNA replication. Our results demonstrate that a mutation in gene 59 of phage T4 causes a premature release of replicative DNA from the replicative complex. This is evidenced by each of the three treatments used to isolate intracellular DNA, namely, lysozyme-Brij 58, lysozyme-Triton X-100, and lysozymeSarkosyl-high temperature. With the use of lysozyme-Triton X-100, 850 S DNA is prematurely released from the >1500 S complex in addition to 63 S DNA. However, only 63 S DNA is released t from the complex when other detergents, such as Brij 58 or Sarkosyl, are used for lysing the infected cells. It is therefore suggested that intracellular DNA in mutantinfected cells is bound by weak electrostatic forces to cell components to form the > 1500 S material. Although there are certain similarities, gene 59 mutants are different in several aspects from mutants in the other two DNA arrested genes, 46 and 47. (1) Host DNA degradation is normal in cells infected with gene 59 mutants (Wu et al., 1972) but not in cells infected w-ith gene 46 or 47 mutants IO 20 30 taO!?oM 2 O 3o (Wiberg, 1966; Kutter and Wiberg, 1968). FRACTION NUMBER (2) Synthesis of gene 59 mutant DNA is FIG. 9. Alkaline sucrose gradient sedimentacompletely arrested, whereas synthetis of tion analysis of intracellular DNA in T4 amC5 and gene 46 and 47 mutant DNA is only parwild-type T4D infected cells. At 3 min after infectially arrested (Wiberg, 1966; Kutter and tion, 3H-thymine (10 FCi/3 fig/ml) and thymine-21968; Yeh, unpublished data.). 1% (1 &i/3 &g/ml) were added to T4 amC5 and Wiberg, (3) Mutation of gene 59 will not cause a T4D infected cells, respectively. Samples from breakdown of the newly synthesized DNA these two infected cultures were mixed and lysed by lysozyme-Triton-NaOH method. Conditions following cessation of DNA synthesis as is for centrifugation were described as in Fig. 10. found in gene 46 and 47 mutants. (4) For e-0, 3H-T4 amC5; C---C, “C-T4D. The gene 59 mutants, inhibition of late protein arrows indicate the position of reference T4 DNA synthesis by chloramphenicol or mutation which was centrifuged in a parallel tube. of gene 33 or 55 not only restores DNA synthesis but also formation of concatemers; lysozyme-Brij 58 method in E. coli pol Ahowever, for gene 46 and 47 mutants, only cells contains lipid, protein, DNA, and DNA DNA synthesis is restored (Shah and synthesizing activity. This DNA synthesizBerger, 1971). ing activity can be partially solubilized with What is the role of gene 59? The attachTriton X-100. Miller and Kozinski (1970) ment of growing points to the membrane found that the > 1500 S material isolated by has been reported in various systems. One the lysozyme-Triton X-100 method in T4 phage-infected cells is formed from a non- may envisage gene 59 as a structural gene specific DNA-protein complex (850 S). The for one of the proteins involved in the attachment of growing points. However, the physical structure and chemical properties observations on the parental and very early of these complexes are not fully understood, 14
II MIN
a
b
15 MIN
1
120
WU AND
C
BOTTOM
FRACTION
YEH
NUMBER
FIG. 10. Sedimentation profile of parental mutant DNA in alkaline sucrose gradient (5 to 20%) Escherichia coli B Tr-201 cells were infected with W-thymine labeled T4D (O---O) or with aH-thymine labeled T4 amC5 (0-O). DNA was extracted by the lysozyme-Triton-NaOH method. Arrows indicate the position of reference T4 DNA.
replicative intermediates do not favor this hypothesis because a normal initiation of DNA replication and formation of a replication complex occurs in gene 59 mutantinfected cells. Furthermore, suppression of the mutant gene 59 phenotype with chloramphenicol (Wu et al., 1972) also suggests that gene 59 does not code for a structural protein. However, we cannot exclude the possibility that the gene 59 protein is involved in attachment of growing points to the membrane in the late replication complexes (e.g., after one round of replication). An alternative explanation of the function of gene 59 is that it is involved in the structural integrity of phage DNA; structural defects in the DNA might cause the premature dissociation of the replication complexes that are observed. Gene 59 mutant-infected cells are characterized by (1) little or no
concatemer formation and, (2) slow repair of fragmented progeny DNA, both of which may affect the stability of replication complexes. Since DNA molecules of phage T4 are circularly permuted and terminally redundant (Thomas and Rubenstein, 1964), the formation of concatameric phage DNA may be involved in creating the circular permutation. The concatemeric DNA can be detected in wild-type infected cells after 7 min infection which is close to the effective time of phenotypic arrested DNA synthesis in gene 59 mutant-infected cells. The concatemers can be formed either by replication of progeny DNA or by recombination or by both mechanisms (Gilbert and Dressler, 1968; Thomas et al., 1968). By the use of computer simulation, Miller et al. (1970), have inferred that the concatemeric DNA
REPLICATIVE
INTERMEDIATES
appearing 15 min after infection are produced as a result of the recombination process of breakage and reunion. If the concatemers are generated by recombination, it is likely that the gene 59 protein is involved in the recombination process, which is consistent with the observations that gene 59 mutants have an increased UV sensitivity and are also defective in the repair of fragmented progeny DNA (Wu and Yeh, to be published). These results favor the hypothesis that gene 59 is involved in the structural integrity of phage DNA. At present, we cannot rule out the possibility that gene 59 protein protects T4 DNA from nucleolytic damage, which can release the DNA from the membrane as broken concatemers incapable of further replication. ACKNOWLEDGMENTS We are grateful to Dr. Irwin Tessman for his stimulating discussions. This work was supported in part by the U.S. Public Health Service research grant GM 18012,and in part by the American Cancer Society. REFERENCES ABELSON, J., and THOMAS, C. A., JR. (1966). The
anatomy of the T5 bacteriophage DNA molecule J. Mol. Biol. 18, 262-291. ADAMS, M. H. (1959). “Bacteriophages”.
Wiley (Interscience), New York. ALTMAN, S., and LERMAN, L. S. (1970). Kinetics and intermediates in the intracellular synthesis of bacteriophage T4 deoxyribonucleic acid. J. Mol. Biol. 50, 235-261. BOLLUM, F. J. (1966). Filter paper disk techniques for assaying radioactive macromolecules. In “Procedures in Nucleic Acid Research” (G. L. Cantoni, and D. R. Davies, eds.), pp. 296-300. Harper & Row, New York. BURGI, E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309-321. Enzymes.” COHEN, S. S. (1968). “Virus-Induced Columbia Univ. Press, New York. DAVERN, C. I. (1971). Molecular aspects of genetic Progr. Nucl. Acid Res. Mol. recombination. Biol. 11, 229-252. DELIUS, H., HOWE, C., and KOZINSKI, A. W. (1971). Structure of the replicating DNA from bacteriophage T4. Proc. Nat. Acad. Sci. U.S. 68, 30493053. DIGGELMANN, H., PULITZER, J. F., and GEIDUSCHEF, E. P. (1970). Relationship between gene 55 function, late transcription and an inter-
OF T4 GENE 59 MUTANTS
121
mediate in the replication of bacteriophage T4 DNA. J. Mol. Biol. 49, 509-514. EARHART, C. F. (1970). The association of host and phage DNA with the membrane of Escherichia
coli. Virology
42, 429-436.
EDGAR, R. S., and WOOD, W. B. (1966). Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells. Proc. Nat. Acad. Sci. U.S. 55, 498-505. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLY, R., EDG.~R, R. S. SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. FR~NKEL, F. R. (1966). Studies on the nature of replicating DNA in T4-infected Escherichia coli. J. Mol. Biol. 18, 127-143. FRANKFIL, F. R. (1968). Evidence for long DNA strands in the replicating pool after T4 infection. Proc. Nat. Acad. Sci. U.S. 59, 131-138. FUCHS, E., and H~NAW~LT, P. (1970). Isolation and characterization of the DNA replication complex from Escherichia coli. J. Mol. Biol. 52, 301-322. GILBERT, W., and DRESSLER, D. (1968). DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33, 473-484. GOULIAN, M. (1971). Biosynthesis of DNA. Anna. Rev. Biochem.
40, 855-898.
GRAY, H. B., and HEARST, J. E. (1968). Flexibility of native DNA from the sedimentation behavior as a function of molecular weight and temperature. J. Mol. Biol. 35, 111-129. JIICOB, F., BRENNER, S., and CUZIN, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329348. KLEIN, A., and BONHOEFFER, F. (1972). DNA replication. Annu. Rev. Biochem. 41,301-322. KNIPPERS, R., and SINSHEIMER, R. L. (1968). Process of infection with bacteriophage +X174. XX. Attachment of the parental DNA of bacteriophage +X174 to a fast-sedimenting cell component. J. Mol. Biol. 34, 17-29. KNIPPERS, R., and STRATLING, W. (1970). The DNA replicating capacity of isolated E. coli cell wall-membrane complexes. Nature (London) 226, 713-717. KOZINSKI, A. W., and LIN, T. H. (1965). Early intracellular events in the replication of T4 phage DNA, I. Complex formation of replicative DNA. Proc. Nat. Acad. Sci. U.S. 54, 273-278. KUTTER, E. M., and WIBERG, J. S. (1968). Degradation of cytosine-containing bacterial and bacteriophage DNA wafter infection of Escherichia co& B with bacteriophage T4D wild type
122
WU AND
and with mutants defective in genes 46, 47 and 56. J. Mol. Biol. 38, 395-411. LITWIN, S., SHAHN, E., and KOZINSICI, A. W. (1969). Interpretation of sucrose gradient sedimentation pattern of deoxyribonculeic acid fragments resulting from random breaks. J. Viral. 4, 24-30. MILLER, R. C., JR., and KOZINSKI, A. W. (1970). Early intracellular events in the replication of bacteriophage T4 deoxyribonucleic acid. V. Further studies on the T4 Protein-deoxyribonucleic acid complex. J. Viral. 5, 490-501. MILLER, R. C., JR., KOZINSKI, A. W., and LITWIN, S. (1970). Molecular recombination in T4 bacteriophage deoxyribonuceleic acid. III. Formation of long single strands during recombination. J. Viral. 5, 368-380. MOSIG, G. (1970). Recombination in bacteriophage T4. Advan. Genet. 15, l-53. MURRAY, R. E., and MBTHE~s, C. K. (1969). Addition of nucleotides to parental DNA early in infection by bacteroiphage T4. J. Mol. Biol. 44, 233-248. ROSENBERG, B. H., and CAVSLIERI, L. F. (1968). Shear sensitivity of the E. coli genome: Multiple membrane attachment points of the E. coli DNA. Cold Spring Harbor Symp. Quant. Biol. 33, 65-72. SALIVAR, W. O., and SINSHEIMER, R. L. (1969). Intracellular location and number of replicating parental DNA molecules of bacteriophages lambda and +X174. J. Mol. Biol. 41, 3965. SHAH, D. B., and BERGER, H. (1971). Replication of gene 46-47 amber mutants of bacteriophage T4D. J. Mol. Biol. 57, 17-34. SHLESER, R., PUGA, A., and TESSMAN, E. (1969). Synthesis of replicative form DNA and m-RNA by gene IV mutants of bacteriophage S13. J. Virol. 4, 394-399. SINSHEIMER, R. L., KNIPPERS, R., and KOMANO,
YEH T. (1968). Stages in the replication of bacteriophage +X174 DNA it/ uivo. Cold Spring Harbor Symp. Qzbant. Biol. 33, 443-448. STRATLING, W., and KNIPPERS, R. (1971). Properties of the DNA synthesizing activity in DNAmembrane complexes from bacterial cell extracts. Eur. J. Biochem. 20, 330-339. SUEOK~, N., and QUINN, W. G. (1968). Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harbor Symp. Quant. Biol. 33, 695705. THOMSS, C. A., and RUBENSTEIN, I. (1964). The arrangements of nucleotide sequences in T2 and T5 bacteriophage DNA molecules. Biophys. J. 4, 93-106. THOMAS, C. A., JR., KELLY, T. J., and RHOADES, M. (1968). The intracellular forms of T7 and P22 DNA molecules. Cold Spring Harbor Symp. Quant. Biol. 33, 417-424. VOGEL, H. J., and BONNER, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 128, 97-106. WERNER, R. (1968). Initiation and propagation of growing points in the DNA of phage T4. Cold Spring Harbor Symp. Quant. Biol. 33, 50-508. WIBERG, J. S. (1966). Mutants of bacteriophage T4 unable to cause breakdown of host DNA. Proc. Nat. Acad. Sci. U.S. 55, 614-621. Wu, R., and YEH, Y. C. (1970). DNA replication in mutants defective in gene 59 to T4 bacteriophage-infected E. coli. Bacterial. Proc., p. 167. WV, R., MA, F. J., and YEH, Y. C. (1972). Suppression of DNA-arrested synthesis in mutants defective in gene 59 of bacteriophage T4. Virology 47, 147-156. YAMAGUCHI, K., MURAKAMI, S., and YOSEIIKAWA, H. (1971). Chromosome-membrane association in Bacillus subtilis. I. DNA release from membrane fraction. Biochem. Biophys. Res. Commun. 44, 1559-1565.
69, 108-122 (1974)
DNA
Arrested
Mutants
of Gene
II. Replicative REEN
WU’
AND
59 of Bacteriophage
T4
Intermediates YUN-CHI
YEH
Department of Biochemistry, University of Arkansas School of Medicine, Little Rock, Arkansas 7%?Oi Accepted January 7, 1974 The abnormal DNA replication of bacteriophage T4 containing amber nonsense mutants in gene 69 (amC5, amHL628) has been studied. Zonal centrifugal analysis of the mutant intracellular DNA molecules isolated from a nonpermissive host, Escherichia coli B, reveals the following: (1) Parental mutant DNA as well as nascent mutant DNA can be recovered by sucrose gradient centrifugation as a fast sedimenting fraction (>1500 S) which is presumably a cell-membrane DNA replication complex. (2) At 2-3 min after initiation of viral DNA synthesis, a gradual accumulation of mutant DNA with a sedimentation coefficient of 63 S is observed. The molecular weight of this DNA is close to, or a little less than, that of a mature viral DNA, suggesting that less than one round of replication occurs before the complete arrest of DNA synthesis. (3) In contrast to what is found in wild-type infected cells, little or no long singlestranded DNA (concatemer) appears in mutant infected cells. Concatenated molecules of DNA, which have been proposed as intermediates in viral replication, are normally found in wild-type infected cells 7 min after infection. (4) Unlike the case of cells infected with mutants of gene 46 and 47, addition of chloramphenicol prevents premature release of the mutant DNA of gene 59 from the fast-sedimenting fraction (>1500 S). INTRODUCTION
Studies of phage T4 DNA synthesis in Escherichia coli have shown that in addition to replication mechanisms, recombination and repair are also involved (Cohen, 1968; Mosig, 1970; DaVern, 1971). These mechanisms are evidenced by the various abnormal phenotypes found among many of the conditional lethal mutants isolated by Epstein et al. (1963) that affect DNA synthesis. A multifork mode of replication occurs in T4-infected cells (Werner, 1968). Recently a bidirectional replication mechanism has also been suggested by electron microscope studies of replicative intermediates (Delius et al., 1971). Physical studies of replicative intermediates (Frankel, 1968; Miller et al., 1 Present address: Department of Biology, University of California, San Diego, La Jolla, California 92037.
1970) revealed that fast sedimenting DNA molecules are associated with the cell membrane, as was observed in various systems including E. coli (Rosenberg and Cavalieri, 1968; Fuchs and Hanawalt, 1970; Stratling and Knippers, 1971), B. subtilis (Sueoka and Quinn, 1968; Yamaguchi et al., 1971), pTX174 (Sinsheimer et al., 1968; Salivar and Sinsheimer, 1969), and S13 (Shleser et al., 1969). Frankel (1968) and Miller et al. (1970) have observed concatemers, replicative intermediates which are present 7 min after infection. They have shown that the strands sediment rapidly because they are concatenates of two or more genomes, not because of an altered conformation of the T4 DNA molecules. We are interested in the mechanisms involved in DNA replication following initiation, and have studied abnormal DNA replicat,ion by mutants of gene 59 (amHL108
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
REPLICATIVE
INTERMEDIATES
628, umC5). The mutants map between the maturation defective (MD) mutant gene 33 and recombination defective mutant gene 32 (Edgar and Wood, 1966). We have reported that the arrested synthesis of DNA caused by mutations in gene 59 can be restored by addition of chloramphenicol or by mutations in gene 55 and 33 (Wu et al., 1972). It is suggested, therefore, that at least one late protein is required for the expression of the mutant gene 59 phenotype. The present work focuses on physicochemical studies of DNA synthesis by umber mutants of gene 59 (amC5, ccmHL628) in the nonpermissive host, E’. coli B. We shall describe the abnormal dissociation of mutant DNA from the membrane-replication complex and also defects in concatemer formation. A possible role of gene 59 in the continuation of DNA synthesis will be discussed. There has been a preliminary account of some of these experiments (Wu and Yeh, 1970). MATERIALS AND METHODS Bacteria and bacteriophage. E. coli B strain Tr201, a thymine-requiring mutant obtained from Dr. G. R. Greenberg, was used as a nonpermissive host for amber phage mutants; E. coli CR63 (Stir+) was the permissive host used for growing the amber mutants. Two T4D amber mutants in gene 59, amHL628 and umC5, were supplied by Dr. R. Edgar. We have purified the mutants genetically by backcrossing twice to wildtype T4D. Also T4 umB272, which is defective in gene 23 (major head protein), was used. A double mutant amC5-amB272 was constructed by genetic recombination. Phage lysates were prepared in liquid culture and purified by differential centrifugation (Adams, 1959). Chemical reagents. All radioisotopes were from the New England Nuclear Company and all commercial enzymes were from the Worthington Biochemical Corporation. Media. Vogel and Banner medium (1956) was used in the isotope labeling experiments. It was made by mixing 0.1 ml. of 20 % glucose, 0.1 ml of 5 % casein hydrolyzate and 10 ml of 1 X salt solution (50 X salt solution was prepared by dissolving 10 g of MgSOd.
OF T4 GENE 59 MUTANTS
109
7HzO. 100 g of citric acid *Hz0 and 500 g each of KZHPOJ and NaNH4HP04. 4Hz0 in 670 ml water). This mixture was named 1 X CT- medium. The medium containing thymine or thymidine (5 pg/ml) was named 1 X CT+. Growth and infection of cells. An overnight culture of E. coli was diluted 50 times with fresh 1 X CT+ medium. The bacteria were grown with vigorous aeration at 37” until the optical density reached 0.8 at 590 nm on a Zeiss spectrophotometer, corresponding to a viable count of about 5 X 108 cells/ml. The cells were centrifuged and resuspended in an equal volume of 1 X CT- medium in the presence of 20 pg/ml D,I,-tryptophan. Phage was added at a multiplicity of approximately 5 phage particles per bacterium. Three minutes after infection, 14C-thymine (1 &J/3 pg/ml) or 3H-methylthymine (10 &i/3 pg/ml) was added. In all experiments, the fraction of bacteria surviving was less than l%, and in most cases, it was considerably less. In labeling the DNA of the parental phage, the cultures of E. coli were infected at a multiplicity of 0.1 phage per bacterium in the presence of 3H-thymine (50 pCi/3 pg/ml) or 14C-thymine (5 &i/l5 pg/ml). After 5 hr of incubation, a few drops of chloroform were added and aeration was continued for an additional 10 min and then the lysate was purified (Adams, 1959). In most cases the labeled phage was used within a week. Treatment of radioactive samples. The radioactivity in acid-insoluble fractions was determined by pipetting 0.1 ml of a sample onto a 2.5 cm disk of Whatman No. 3 filter paper. The disks were washed for 10 min in three successive 209ml volumes of 5% trichloroacetic acid (TCA) followed by two successive rinses in 200 ml of 95 % alcohol. This is a slight modification of the procedure described by Bollum (1966). The filters were then dried and placed in vials containing 5 ml each of toluene phosphor (4g/l PPO and 50 mg/l POPOP) and counted in a Nuclear Chicago liquid scintillation counter. Sucrose gradients and centrifugation. For neutral sucrose gradients, 5 ml linear 5 to 20 % sucrose gradients were layered over 1 ml saturated sucrose. Sucrose solutions were
110
WU ilND
prepared in 1 X SSC solution (0.15 N NaCl and 0.015 N sodium citrate, pH 7.5) in the presence of 0.015 n/r EDTA. For alkaline sucrose gradients, linear 5 to 20 % sucrose gradients were also layered over 1 ml of saturated sucrose in order to improve sample recovery. Alkaline sucrose solutions were prepared in 0.05 M NasP04 buffer (pH 12.2) in the presence of 0.015 M EDTA. Centrifugation was performed at 25,000 rpm for 30 min and 30,000 rpm for 60 min for neutral and alkaline gradients, respectively, in a SW 40 rotor of a Beckman Model L2-65B preparative ultracentrifuge. The tubes were pierced and 8 drops per fraction were collected onto Whatman No. 3 filter papers. After drying, the radioactivity of the acid-insoluble fractions was assayed. Lysing procedures. The procedure of Frankel (1966) was used for lysing cells before neutral sucrose gradient centrifugation. Cells were pipetted into an equal volume of a solution consisting of 0.1 M EDTA, 0.1 M NaCN, and 200 pg egg white lysozyme/ml at pH 8.5, and the mixture was placed in a 65” water bath. Partial clearing occurred after 1 min, at which time sarkosyl NL 97 was added to 0.1%. Incubation was continued for an additional 10 min and the resultant clear lysate was removed from the water bath and cooled to room temperature. In preparation for alkaline sucrose gradient centrifugation, we used two lysing procedures, one described by Frankel (1968) and another by Miller et al. (1970). In the former method the cells are treated with 1 mg lysozyme/ml, 3% sarkosyl NL 97 (Geigy Industrial Chemicals) and 0.2 N NaOH. In the latter method the cells are cooled in an ice bath and then treated with 100 pg lysozyme/ml for 10 min, after which Triton X-100 is added to a concentration of 1 %, and 10 min later the DNA is denatured by addition of NaOH to 0.4 N. In our hands, the lysozyme-Triton method caused less hydrodynamic shearing than the method described by Frankel. Isolation of replication complex. Two methods were used in this study. One was adapted from Miller and Kozinski (1970), and was similar to the lysozyme-TritonNaOH method, except that NaOH was not
YEH
added and the Triton X-100 treatment was prolonged to 4 hr. The other was a slight’ modification of Knippers and Sinsheimer’s method (1968). Infected cells (5 X 108 cells) were suspended in 0.25 ml. of 20% sucrose solution (0.05 &! Tris, 0.05 1M NaCl, 0.05 M EDTA, pH 8.0) ; 0.05 ml of 2 mg lysozyme/ ml was added, and the mixture was incubated for 20 min at 37”. Next., 0.2 ml of 5 % Brij 58 (Atlas Chemical Industries, Inc.) was added, and incubation continued for 10 more min. Finally, 0.5 ml of the same TrisNaCl-EDTA solution was added, followed by gentle mixing. This method will be designated lysozyme-Brij method. CaZcuZation. The data from sucrose gradient centrifugation were analyzed by the use of a computer program. Molecular weights and sedimentation coefficients were calculated according to t’he equation of Burgi and Hershey (1963). The number of nicks in the chromosome was estimated using the method of Litwin et al. (1969). The molecular weight of T4 standard DNA was taken as 118.3 X lo6 for double-stranded DNA (Gray and Hearst, 1968). The s value of T4 mature DNA is taken as 63 S (Frankel, 1966). RESULTS
Awested DNA Synthesis After the initiation of DNA synthesis by amber mutants of gene 59 (amC5, amHL628) in the nonpermissive host E. coli B, it is not known whether synthesis is completely stopped or a steady state of synthesis and degradation of DNA is maintained. The kinetics of DNA synthesis in E. coli B (thy-) infected with T4 am+ and T4 amC.5 (gene 59) were studied by pulse-labeling with 3H-methylthymine into acid-insoluble material. The results (Fig. 1) show that initially the mutant synthesizes DNA at the same increasing rate as the wild-type, but it reaches its maximum rate of incorporation at about 7-8 min after infection following which the rate decreases rapidly. In contrast, wild-type infected cells show a rate of synthesis that increases to a high plateau level. This result, and also the one obtained by continuous labeling (Fig. I), clearly indicate that the observed arrest of DNA synthesis is not due to a steady state caused
REPLICATIVE
TIME
AFTER
INFECTION
INTERMEDIATES
(MIN)
FIG. 1. Kinetics of DNA synthesis in cells infected with wild-type T4D and T4 amC5 (gene 59). Thymine-2-W (1 &i/3 pg/ml) was added at the beginning of infection. At various times, 0.2 ml of infected culture was pulse labeled for 30 set with ZH-thymine (20 &i/O.14 rg). The incorporation was stopped by adding an equal volume of cold 0.1 M KCN-tJ.05 EDTA pH 8.0. Samples of 0.1 ml were taken to measure radioactivity in the acidinsoluble fraction as described in Materials and Methods. (O), 3H-thymine; (0), thymine-2-W incorporated into T4 amC5 infected cells. (A), 3H-thymine; (a) thymine-2-14C incorporated into T4D infected cells.
by synthesis and degradation. It is seen that the mutation in gene 59 has an effect by 2-3 min after DNA synthesis is initiated and eventually causes a complete arrest of DNA synthesis. Furthermore, the newly synthesized mutant DNA is quite stable with no detectable degradation. Premature Release of Mutant Replication Complex
DNA
from
Replicative intermediates in Brij-58 treated lysates. The involvement of a DNA-protein complex has been demonstrated in T4 phage replicative cycles (Kozinski and Lin, 1965). The complex is thought to be attached to the cell membrane and is frequently referred to as a “membrane” complex (Frankel, 1968; Altman and Lerman, 1970; Earhart, 1970). Replicative intermediates of T4 amC5 in-
OF T4 GENE 59 MUTANTS
111
fected cells have been studied by using nonionic detergents (Brij 58 or Triton X-100) for lysing the infected cells and analyzing in neutral sucrose gradient sedimentation. When lysozyme-Brij 58 is used for lysing medium immediately after pulselabeling (Fig. 2a, f), most of newly labeled DNA (from 4.5 to 5 min after infection) in both T4 am+ and am- infected cells sediment faster than 1500 S and can be recovered from the cushion; the exact s value cannot be measured. Other investigators have found in T4-infected cells a similar complex with reports of sedimentation values up to ~800 S, even though the isolation procedures were somewhat different (Altman and Lerman, 1970; Earhart, 1970; Miller and Kozinski 1970; Shah and Berger, 1971). This fast-sedimenting property indicates that a complex of labeled DNA with proteins or membrane is formed. Otherwise, the sedimentation value of replicative DNA would be smaller. The premature release of the mutant DNA from the replication complex is evidenced by the following. About 24% of 3H-labeled DNA in T4 amC5 infected cells obtained after a 2.5-min chase (7.5 min after infection) is released from the complex (> 1500 S) and therefore remains close to the top of gradient (Fig. 2b). This slowly sedimenting material is found close to the top of gradient (Fig. 2b). This slowly sedimenting material is found close to the position of mature T4 DNA (63 S). The fraction of this prematurely released DNA (63 S) increases as infection continues. About 35 % and 40% of the labeled DNA are released from the complex (> 1500 S) 9.5 and 15 min after infection, respectively, in mutant-infected cells (Fig. 2c-d); then a drop to 34% occurs at 30 min (Fig. 2e). However, this premature release of DNA from the fast-sedimenting complex is not observed throughout the replicative cycle in wild-type infected cells (Fig. Zf-j). ’ In addition to this 63 S material, a second peak of DNA appears in mutant-infected cells 15 min after infection (Fig. 2d). It sediments as rapidly as 14C-labeled phage particles (-800 S) and resists DNase digestion. This property suggests that the 800 S
WU AND YEH
112 T4mC5
T4D
40 20 0 40 20 0 4BOTTOM
FRACTION
NUMBER
FIG. 2. Neutral sucrose gradient analysis of intracellular DNA lysed by lysozyme-Brij 58 method. Escherichia coli B Tr201 cells were infected with T4D or T4 czmC5. *H-Thymine (20 &i/ml) was added at 4.5-5.5 min after infection. The pulse was terminated by the addition of an equal volume of medium containing
thymine
(2 mg/ml).
Samples of infected
cells at various
times after infection
were lysed by the
lysozyme-Brij 58 method in the presence of ‘Glabeled T4 phage. Samples were centrifuged for 30 min at 10,OCOrpm as described under Materials and Methods. (a) to (e) were DNA extracted from cells infected with T4 a&5. (f) to (j) were DNA extracted from cells infected with T4D. (h) and (i) were independent experiments from the rest. The sedimentation is from right to left in this and subsequent figures. a---@, aH-labeled DNA; O---O, ‘“C-labeled reference T4 DNA. DNA is a mature phage particle, and indicates that maturation occurred even in the mutant phage. As shown in Fig. 2 (e and j) 41% and 71% of 3H-labeled DNA are encapsulated into phage particles after 30 min in mutantand wild-type infected cells, respectively. This observation is consistent with the burst size in E. coli B of this mutant phage of 10 to 15 infective centers/ bacterium.
This dissociation of the replicative complex formed by the newly synthesized DNA suggests that the product of gene 59 is required for stabilizing the replication complex. In order to investigate whether the premature release of T4 amC5 DNA from the membrane complex is due to the instability of the complex or due to encapsulation, an amber mutation in gene 23 (head protein)
REPLICATIVE
INTERMEDIATES
was introduced by genetic crossing. DNA encapsulation is inhibited in this double mutant (T4 amC5 -amB272, gene 59-23) ; however, the premature release phenomenon is still observed in mutant-phage-infected cells (Fig. 3). In addition, 90% of the amB272 DNA still remains in the fast sedimenting complex (2 1500 S). This result shows that the maturation process is not involved in the expression of premature DNA release. Furthermore, it is possible to eliminate the mature phage progeny from the replicative intermediates. Replicative intermediates in Triton X-100 T4
OF T4 GENE
59 MUTANTS
113
treated lysates. To obtain further information about replication complexes and premature DNA release, another nonionic detergent, Triton X-100, is used to isolate the replication complex from mutant-phageinfected cells. The experiment, similar to that of Fig. 2, is carried out in cells infected with the double mutant amC5-amB272. Immediately after a short isotope pulse (4.5-5.5 min after infection), most of the labeled replicative DNA sediments as rapidly as the complex (> 1500 S) isolated by the lysozyme-Brij 58 method (Fig. 4a). After a 2.5 min chase (7.5 min after infection), the
amC5-am8272 -amC5-018272
14 amB272 -
t
BOTTOM
FRACTION
NUMBER
FIG. 3. Neutral sucrose gradient sedimentation of intracellular DNA lysed by lysozyme Brij 58 method. Escherichia coli B Tr291 was infected with T4 amC5-amB272 (gene 59-23) or T4 amB272 (gene 23, used as a control). Conditions of isotope labeling and centrifugation were as described in Fig. 2. Cells were lysed by the lysozyme-Brij 58 method in the presence of ~KXabeled reference T4 DNA. In (a) to (d), DNA was extracted from T4 amC5-amB272 infected cells. In (e) and (f) DNA was extracted from 0, 8H-Labeled intracellular DNA; O-O, “C-labeled reference T4 T4 amB272 infected cells. l DNA.
114
WU AND YEH T4
a~C5-0~~1B272
w a
T4 amB27
f
2
w
BOTTOM
FRACTION
NUMBER
FIG. 4. Neutral sucrose gradient sedimentation of intracellular DNA lysed by the lysozyme-Triton X-100 method. Intracellular DNA was labeled (4.5-5.5 min after infection) and chased as described in Fig. 2. DNA was isolated by the lysozyme-Triton X-100 method in the presence of ‘%-labeled reference T4 DNA. In (a) to (c), DNA was extracted from T4 amC5amB272 infected cells. In (d), DNA was extracted from T4 amB272 infected cells. O--e, aH-labeled intracellular DNA: O-0, r*C-T4 reference DNA.
labeled 1500 S complex is dissociated into 850 S DNA and 63 S DNA (Fig. 4b). Though the 850 S material sediments close to the position of mature phage DNA particles, it is not the mature phage because of its sensitivity to DNase digestion. Finally, almost 88 % of the labeled DNA was released from the fast-sedimenting complex after 30 min of infection (Fig. 4~). In contrast, DNA from cells infected with umB272 phage sediments faster than 1500 S (Fig. 4d). These results are consistent with those obtained with the Brij 58 procedure as described in the preceding section. Both procedures gave the same gradual accumulation of 63 S DNA 2-3 min after the initiation of replication. Effect of chloramphenicol on premature release of mutant DNA. Previously, we
showed that chloramphenicol (CM) added 5-12 min after infection rapidly restored DNA synthesis, which is otherwise arrested after 12-14 min (Wu et al., 1972). The effect of CM on the premature release of mutant DNA was investigated. Chloramphenicol was added at set intervals to cells infected with the double mutant amC5-amB272. After 30 min the intracellular DNA was isolated by the lysozyme-Brij 58 procedure. Figure 5 shows the effect of chloramphenicol on the formation of the replication complex. It is seen that the addition of chloramphenico1 prior to 7.5 min after infection prevents premature release of mutant DNA from the replication complex (> 1500 S). However, when chloramphenicol is added 10 min after infection (Fig. 5c), 27 % of the replicative DNA is released. Since chloramphenicol is known to inhibit protein synthesis, it is sug-
REPLICATIVE
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FRACTION
OF T4 GENE 59 MUTANTS
115
NUMBER
FIG. 5. Effect of chloramphenicol on the dissociation of the replication complex in T4 amC5 infected cells. Escherichia coli B Tr201 was infected with T4 amB272 or T4 amC5-amB272. Intracellular DNA was labeled at 4.5 to 5.5 min after infection as described in Fig. 2. Chloramphenicol (lOOpg/ml) was added at various times after infection and the cells were lysed 30 min after treatment. The sedimentation was carried out in a 5 to 200%neutral sucrose gradient. Arrows indicate the position of reference T4 DNA.
gested that at least one protein, synthesized beginning approximately 7.5 min after infection, participates in the dissociation of the replication complex in cells infected with gene 59 mutants. Furthermore, the restoration of DNA synthesis by chloramphenicol as well as formation of a stable replicative complex suggests that the fast sedimenting complex (> 1500 S) is essential for continuous viral DNA replication. Absence of Concatemer Formation To determine further the function of gene 59, we have studied the replicative intermediates of DNA molecules which are longer than one phage DNA equivalent unit. Two kinds of DNA molecules that are longer than one phage equivalent unit have been found in T4 phage-infected cells (Frankel, 1968; Murray and Mathews, 1969). One is formed within the first 5 min after infection, which is just before normal replication has commenced (Murray and Mathews, 1969). The other kind, originally described by Frankel (1968), is detectable 7 min after infection. The latter has been named “concatemer” and consists of several DNA molecules joined covalently end to end. Neutral sucrose gradient sedimentation. Figure 6 shows the sedimentation profile of mutant phage intracellular DNA in neutral
sucrose gradients. E. coli B Tr201 was infected with the double mutant amC5anB272. Phage DNA was labeled at the onset of DNA synthesis (from 3.5 min to 4.5 min infection at 37°C) with 3H-thymine, followed by a chase with cold thymine (1 mg/ml). Immediately after pulse-labeling, the newly synthesized DNA in mutantinfected cells sediments close to the reference T4 DNA (63 S) (Fig. 6a). It is very difficult to observe the difference in position between this intracellular DNA and the reference DNA, but occasionally there is a difference of one fraction in the gradient. This material corresponds to the long DNA described by Murray and Mathews (1969) that is 6 % longer than mature T4 DNA. To date, no mutant has been found defective in the synthesis of this long DNA (Murray and Mathews, 1969). At 6 min after infection (1.5 min chase), the labeled DNA sediments faster at a position that corresponds to 200 S material (Fig. 6b). This 200 S DNA, is the proposed precursor of mature phage DNA in wild-type T4-infected cells (Frankel, 1968). However, this fast sedimenting material (200 S) which appears in the early stage of the replicative cycle is transient in mutant infected cells. At 7.5 min after infection, 38 % of 3H-labeled DNA is released from 200 S DNA and has a sedi-
116
WU AND YEH T4amC5-amB272 al 4.5rnl”
---BUTTOM
T4amB272 t
FRACTION
b) 6 Omw
g)6.0msn
NUMBER
FIG. 6. Neutral sucrose gradient sedimentation of the mutant mtracellular DNA lysed by the lysoayme-sarkosyl-high temperature method. Escherichia coli B cells were infected with T4 amCbamB272 or amB272 as a control. *H-Thymine (25 &i/ml) was added at 3.5 min after infection and the incorporation was terminated by the addition of thymine (1 mg/ml) as described in Fig. 2. Intracellular DNA was isolated at various times after infection by the method described by Frankel (1966). Centrifugation was carried out in a SW 40 rotor at aspeed of B,ooO rpm ior 3Umin through & 5 to f20ya neutral swrrxse gradient. O-0, aH-labeled DNA; O-O, 14C-labeled reference T4 DNA.
mentation value similar to the marker T4 DNA (63 S, Fig. 6~). The fraction of 63 S DNA gradually increases until at 13 min after infection (Fig. &), most pulse labeled DNA appears near 63 S position, and this sedimentation profile does not change further (Fig. Sf). In contrast, DNA from cells infected with umB272 phage sediments at 200 S without any release of 63 S fraction during the replicative cycle (Fig. 6g-i). Although it appears that two kinds of replicative structures are generated in mutant-infected cells, one at 63 S and the other at 200 S, a pulse-chase experiment at
7.5 min indicates that the 63 S DNA is produced early in the replicative cycle as a dissociation product of the 200 S DNA (Fig. 7). This observation and the results shown in Fig, 6 suggest that one or more steps in the formation of 200 S DNA is defective in this mutant. Alkaline sucrose gradient sedimentation. Long strands of phage DNA have been observed in alkaline sucrose gradient (Frankel, 1968; MilIer et al., 1970). We have used the lysozyme-Sarkosyl-NaOH method to estimate the rate of joining of fragmented DNA and the size of replicative DNA in mutant-
REPLICATIVE
INTERMEDIATES 635
i
i.
.L IO
20
tBOTTOM
FRACTION
~ 30
40
NUMBER
FIG. 7. The fate of 290 S DNA in T4 amC5 infected cells. At 7 min after infection, mutant infected cells were pulse labeled with XI &i/ml for *H-thymine for 30 see (0), then chased with thymine (1 mg/ml) for 10 min (0). The intracellular DNA was isolated by lysozyme-salkosyl-high temperature method and sedimented through a 5 to 20y0 neutral sucrose gradient as described in Fig. 6. The arrow indicates the position of reference T4 DNA in a parallel tube.
and wild type-infected cells. Almost 50 % of the labeled DNA isolated immediately after pulse labeling (4 to 5.5 min) sediments in alkaline sucrose gradient slower than the position of 0.55 of reference T4 DNA (&‘D?) (Fig. Sa, e). This value corresponds to a molecular weight of about 13 million daltons (Abelson and Thomas, 1966). The fragmented DNA appears to be gradually joined. The second peak (Dz/D~ = 0.7) of DNA fragments is observed after 7.5 min infection in both types of phage-infected cells (Fig. 8b, f). This DNA of molecular weight 25 million is continuously joined in the wild type-infected cells until, at 12 min 2 DZ equals the distance from the top of gradient of the replicative DNA and DI equals the distance from top of the gradient of the standard phage DNA molecules.
OF T4 GENE 59 MUTANTS
117
after infection, most of the labeled DNA sediments faster or at the same rate as reference T4 DNA (Fig. Sh). In contrast to these results, the joining of fragmented DNA is slow in the mutant; after 12 min infection, 30 % of the labeled DNA was still in the position corresponding to Dp/Dl = 0.7 (Fig. Sd). In a separate experiment, the same result was obtained showing the joining of fragmented DNA (corresponding to 25 million daltons) is slow in mutant-infected cells. For comparison, wild-type phage DNA was continuously labeled by 2J4C-thymine and mutant DNA was labeled by 3H-thymine after infection. Samples from these two cultures were mixed and lysed by the lysozyme-Triton X-lOO-NaOH method. At 11 min and 15 min after infection, one-third of the replicative DNA in the mutant- and wild type-infected cells sedimented to a position corresponding to 25 million molecular weight (DJDI = 0.7, Fig. 9a, b). For wild-type phage this fragmented DNA is joined rapidly, only 16 % and 10% of this DNA being left after incubtation for 20 min and 30 min, respectively (Fig. 9c, d). In comparison, 22% of mutant DNA remains fragmented even 30 min after infection (Fig. 9d). This slow joining is consistent with the UV sensitivity found in the gene 59 mutants in which these mutants cannot repair damaged DNA caused by UV irradiation (Wu and Yeh, to be published). In addition to the defect in repairing fragmented DNA either after UV-irradiation or after normal infection with the mutant, there is an obvious difference in the formation of single-stranded DNA longer than one unit length of T4 DNA (the concatemer form). This form can be detected in wild-type infected cells 7.5 min after infection (Fig. Sg, h and 9) DNA twice as long as the mature phage DNA (DJDI = 1.29) is barely detected in mutant replicative cycles using either the lysozyme-SarkosylNaOH method (Fig. 8) or the lysozymeTriton X-160 method (Fig. 9). This observation corresponds well with the neutral sucrose condition in which DNA synthesized in this mutant has shorter strands during formation of the replicative cycle.
118
WU AND YEH
t
BOTTOM
FRACTION
NUMBER
FIG. 8. Alkaline sucrose gradient sedimentation of intracellular DNA lysed by the lysozyme-sarkosyl-NaOH Method. Intracellular DNA in phage-infected cells was labeled with 3H-thymine (2Q&X/ml, 4.5-5.5 min after infection) and chased with thymine (1 mg/ml). Sedimentation was carried out at a speed of 30,000 rpm for 60 min through a 5 to 20% alkaline sucrose gradient. In (a) to (d), DNA was extracted from cells infected with T4 amC5 at various times after infection. In (e) to (h), DNA was extracted from cells infected with T4D. The arrow indicates the position of 0.7 (02/01), except in (a) and (e), which show the position of 0.55 (Dt/Dl). +--0, 3H-labeled DNA; O-O, “C-labeled reference T4 DNA.
This result leads us to conclude that the gene 59 mutant is defective in the formation of long-chain concatemers. Integrity of Parental Mutant Phuge DNA The inability to detect single chains longer than the marker T4 DNA, as well as the slow repairing of fragmented DNA in the mutant, can be explained if, at early times after infection, discontinuities are introduced into the parental template molecules and replication occurs on partially degraded templates. Therefore, the integrity of parental DNA was investigated at early times after infection. It is clear that the parental DNA, after both mutant and wildtype infection, have similar sedimentation profiles, except in the formation of longstranded DNA, which is barely observable in the mutant-infected cells (Fig. 10). However, most of mutant parental DNA has the same sedimentation rate as reference T4 DNA. Therefore, it is concluded that in the absence of gene 59 function, early break-
down of the template molecules does not appear to account for its inability to synthesize long, single-stranded DNA and to repair fragmented DNA. DISCUSSION
Since its first proposal (Jacob et al., 1963), association of replicative DNA with a cell component, presumably the cell membrane, has been reported in many species including prokaryotic as well as eukaryotic cells (Goulian, 1971; Klein and Bonhoeffer, 1972). However, the nature and role of membrane components in DNA synthesis have not been elucidated. Using two nonionic detergents, Brij 58 and Triton X-100, we have shown that a replication complex (> 1500 S) is involved in phage T4 replication. Similar results have been reported (Altman and Lerman, 1970; Diggelmann et aE., 1970; Miller and Kozinski, 1970; Shah and Berger, 1971). Stratling and Knippers (1971) found that this complex (> 1500 S) isolated by the
REPLICATIVE
INTER.MEDIATES
OF T4 GENE 59 MUTANTS
119
but it is evident that they play an important role in T4 DNA replication. Our results demonstrate that a mutation in gene 59 of phage T4 causes a premature release of replicative DNA from the replicative complex. This is evidenced by each of the three treatments used to isolate intracellular DNA, namely, lysozyme-Brij 58, lysozyme-Triton X-100, and lysozymeSarkosyl-high temperature. With the use of lysozyme-Triton X-100, 850 S DNA is prematurely released from the >1500 S complex in addition to 63 S DNA. However, only 63 S DNA is released t from the complex when other detergents, such as Brij 58 or Sarkosyl, are used for lysing the infected cells. It is therefore suggested that intracellular DNA in mutantinfected cells is bound by weak electrostatic forces to cell components to form the > 1500 S material. Although there are certain similarities, gene 59 mutants are different in several aspects from mutants in the other two DNA arrested genes, 46 and 47. (1) Host DNA degradation is normal in cells infected with gene 59 mutants (Wu et al., 1972) but not in cells infected w-ith gene 46 or 47 mutants IO 20 30 taO!?oM 2 O 3o (Wiberg, 1966; Kutter and Wiberg, 1968). FRACTION NUMBER (2) Synthesis of gene 59 mutant DNA is FIG. 9. Alkaline sucrose gradient sedimentacompletely arrested, whereas synthetis of tion analysis of intracellular DNA in T4 amC5 and gene 46 and 47 mutant DNA is only parwild-type T4D infected cells. At 3 min after infectially arrested (Wiberg, 1966; Kutter and tion, 3H-thymine (10 FCi/3 fig/ml) and thymine-21968; Yeh, unpublished data.). 1% (1 &i/3 &g/ml) were added to T4 amC5 and Wiberg, (3) Mutation of gene 59 will not cause a T4D infected cells, respectively. Samples from breakdown of the newly synthesized DNA these two infected cultures were mixed and lysed by lysozyme-Triton-NaOH method. Conditions following cessation of DNA synthesis as is for centrifugation were described as in Fig. 10. found in gene 46 and 47 mutants. (4) For e-0, 3H-T4 amC5; C---C, “C-T4D. The gene 59 mutants, inhibition of late protein arrows indicate the position of reference T4 DNA synthesis by chloramphenicol or mutation which was centrifuged in a parallel tube. of gene 33 or 55 not only restores DNA synthesis but also formation of concatemers; lysozyme-Brij 58 method in E. coli pol Ahowever, for gene 46 and 47 mutants, only cells contains lipid, protein, DNA, and DNA DNA synthesis is restored (Shah and synthesizing activity. This DNA synthesizBerger, 1971). ing activity can be partially solubilized with What is the role of gene 59? The attachTriton X-100. Miller and Kozinski (1970) ment of growing points to the membrane found that the > 1500 S material isolated by has been reported in various systems. One the lysozyme-Triton X-100 method in T4 phage-infected cells is formed from a non- may envisage gene 59 as a structural gene specific DNA-protein complex (850 S). The for one of the proteins involved in the attachment of growing points. However, the physical structure and chemical properties observations on the parental and very early of these complexes are not fully understood, 14
II MIN
a
b
15 MIN
1
120
WU AND
C
BOTTOM
FRACTION
YEH
NUMBER
FIG. 10. Sedimentation profile of parental mutant DNA in alkaline sucrose gradient (5 to 20%) Escherichia coli B Tr-201 cells were infected with W-thymine labeled T4D (O---O) or with aH-thymine labeled T4 amC5 (0-O). DNA was extracted by the lysozyme-Triton-NaOH method. Arrows indicate the position of reference T4 DNA.
replicative intermediates do not favor this hypothesis because a normal initiation of DNA replication and formation of a replication complex occurs in gene 59 mutantinfected cells. Furthermore, suppression of the mutant gene 59 phenotype with chloramphenicol (Wu et al., 1972) also suggests that gene 59 does not code for a structural protein. However, we cannot exclude the possibility that the gene 59 protein is involved in attachment of growing points to the membrane in the late replication complexes (e.g., after one round of replication). An alternative explanation of the function of gene 59 is that it is involved in the structural integrity of phage DNA; structural defects in the DNA might cause the premature dissociation of the replication complexes that are observed. Gene 59 mutant-infected cells are characterized by (1) little or no
concatemer formation and, (2) slow repair of fragmented progeny DNA, both of which may affect the stability of replication complexes. Since DNA molecules of phage T4 are circularly permuted and terminally redundant (Thomas and Rubenstein, 1964), the formation of concatameric phage DNA may be involved in creating the circular permutation. The concatemeric DNA can be detected in wild-type infected cells after 7 min infection which is close to the effective time of phenotypic arrested DNA synthesis in gene 59 mutant-infected cells. The concatemers can be formed either by replication of progeny DNA or by recombination or by both mechanisms (Gilbert and Dressler, 1968; Thomas et al., 1968). By the use of computer simulation, Miller et al. (1970), have inferred that the concatemeric DNA
REPLICATIVE
INTERMEDIATES
appearing 15 min after infection are produced as a result of the recombination process of breakage and reunion. If the concatemers are generated by recombination, it is likely that the gene 59 protein is involved in the recombination process, which is consistent with the observations that gene 59 mutants have an increased UV sensitivity and are also defective in the repair of fragmented progeny DNA (Wu and Yeh, to be published). These results favor the hypothesis that gene 59 is involved in the structural integrity of phage DNA. At present, we cannot rule out the possibility that gene 59 protein protects T4 DNA from nucleolytic damage, which can release the DNA from the membrane as broken concatemers incapable of further replication. ACKNOWLEDGMENTS We are grateful to Dr. Irwin Tessman for his stimulating discussions. This work was supported in part by the U.S. Public Health Service research grant GM 18012,and in part by the American Cancer Society. REFERENCES ABELSON, J., and THOMAS, C. A., JR. (1966). The
anatomy of the T5 bacteriophage DNA molecule J. Mol. Biol. 18, 262-291. ADAMS, M. H. (1959). “Bacteriophages”.
Wiley (Interscience), New York. ALTMAN, S., and LERMAN, L. S. (1970). Kinetics and intermediates in the intracellular synthesis of bacteriophage T4 deoxyribonucleic acid. J. Mol. Biol. 50, 235-261. BOLLUM, F. J. (1966). Filter paper disk techniques for assaying radioactive macromolecules. In “Procedures in Nucleic Acid Research” (G. L. Cantoni, and D. R. Davies, eds.), pp. 296-300. Harper & Row, New York. BURGI, E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309-321. Enzymes.” COHEN, S. S. (1968). “Virus-Induced Columbia Univ. Press, New York. DAVERN, C. I. (1971). Molecular aspects of genetic Progr. Nucl. Acid Res. Mol. recombination. Biol. 11, 229-252. DELIUS, H., HOWE, C., and KOZINSKI, A. W. (1971). Structure of the replicating DNA from bacteriophage T4. Proc. Nat. Acad. Sci. U.S. 68, 30493053. DIGGELMANN, H., PULITZER, J. F., and GEIDUSCHEF, E. P. (1970). Relationship between gene 55 function, late transcription and an inter-
OF T4 GENE 59 MUTANTS
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mediate in the replication of bacteriophage T4 DNA. J. Mol. Biol. 49, 509-514. EARHART, C. F. (1970). The association of host and phage DNA with the membrane of Escherichia
coli. Virology
42, 429-436.
EDGAR, R. S., and WOOD, W. B. (1966). Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells. Proc. Nat. Acad. Sci. U.S. 55, 498-505. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLY, R., EDG.~R, R. S. SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. FR~NKEL, F. R. (1966). Studies on the nature of replicating DNA in T4-infected Escherichia coli. J. Mol. Biol. 18, 127-143. FRANKFIL, F. R. (1968). Evidence for long DNA strands in the replicating pool after T4 infection. Proc. Nat. Acad. Sci. U.S. 59, 131-138. FUCHS, E., and H~NAW~LT, P. (1970). Isolation and characterization of the DNA replication complex from Escherichia coli. J. Mol. Biol. 52, 301-322. GILBERT, W., and DRESSLER, D. (1968). DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33, 473-484. GOULIAN, M. (1971). Biosynthesis of DNA. Anna. Rev. Biochem.
40, 855-898.
GRAY, H. B., and HEARST, J. E. (1968). Flexibility of native DNA from the sedimentation behavior as a function of molecular weight and temperature. J. Mol. Biol. 35, 111-129. JIICOB, F., BRENNER, S., and CUZIN, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329348. KLEIN, A., and BONHOEFFER, F. (1972). DNA replication. Annu. Rev. Biochem. 41,301-322. KNIPPERS, R., and SINSHEIMER, R. L. (1968). Process of infection with bacteriophage +X174. XX. Attachment of the parental DNA of bacteriophage +X174 to a fast-sedimenting cell component. J. Mol. Biol. 34, 17-29. KNIPPERS, R., and STRATLING, W. (1970). The DNA replicating capacity of isolated E. coli cell wall-membrane complexes. Nature (London) 226, 713-717. KOZINSKI, A. W., and LIN, T. H. (1965). Early intracellular events in the replication of T4 phage DNA, I. Complex formation of replicative DNA. Proc. Nat. Acad. Sci. U.S. 54, 273-278. KUTTER, E. M., and WIBERG, J. S. (1968). Degradation of cytosine-containing bacterial and bacteriophage DNA wafter infection of Escherichia co& B with bacteriophage T4D wild type
122
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and with mutants defective in genes 46, 47 and 56. J. Mol. Biol. 38, 395-411. LITWIN, S., SHAHN, E., and KOZINSICI, A. W. (1969). Interpretation of sucrose gradient sedimentation pattern of deoxyribonculeic acid fragments resulting from random breaks. J. Viral. 4, 24-30. MILLER, R. C., JR., and KOZINSKI, A. W. (1970). Early intracellular events in the replication of bacteriophage T4 deoxyribonucleic acid. V. Further studies on the T4 Protein-deoxyribonucleic acid complex. J. Viral. 5, 490-501. MILLER, R. C., JR., KOZINSKI, A. W., and LITWIN, S. (1970). Molecular recombination in T4 bacteriophage deoxyribonuceleic acid. III. Formation of long single strands during recombination. J. Viral. 5, 368-380. MOSIG, G. (1970). Recombination in bacteriophage T4. Advan. Genet. 15, l-53. MURRAY, R. E., and MBTHE~s, C. K. (1969). Addition of nucleotides to parental DNA early in infection by bacteroiphage T4. J. Mol. Biol. 44, 233-248. ROSENBERG, B. H., and CAVSLIERI, L. F. (1968). Shear sensitivity of the E. coli genome: Multiple membrane attachment points of the E. coli DNA. Cold Spring Harbor Symp. Quant. Biol. 33, 65-72. SALIVAR, W. O., and SINSHEIMER, R. L. (1969). Intracellular location and number of replicating parental DNA molecules of bacteriophages lambda and +X174. J. Mol. Biol. 41, 3965. SHAH, D. B., and BERGER, H. (1971). Replication of gene 46-47 amber mutants of bacteriophage T4D. J. Mol. Biol. 57, 17-34. SHLESER, R., PUGA, A., and TESSMAN, E. (1969). Synthesis of replicative form DNA and m-RNA by gene IV mutants of bacteriophage S13. J. Virol. 4, 394-399. SINSHEIMER, R. L., KNIPPERS, R., and KOMANO,
YEH T. (1968). Stages in the replication of bacteriophage +X174 DNA it/ uivo. Cold Spring Harbor Symp. Qzbant. Biol. 33, 443-448. STRATLING, W., and KNIPPERS, R. (1971). Properties of the DNA synthesizing activity in DNAmembrane complexes from bacterial cell extracts. Eur. J. Biochem. 20, 330-339. SUEOK~, N., and QUINN, W. G. (1968). Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harbor Symp. Quant. Biol. 33, 695705. THOMSS, C. A., and RUBENSTEIN, I. (1964). The arrangements of nucleotide sequences in T2 and T5 bacteriophage DNA molecules. Biophys. J. 4, 93-106. THOMAS, C. A., JR., KELLY, T. J., and RHOADES, M. (1968). The intracellular forms of T7 and P22 DNA molecules. Cold Spring Harbor Symp. Quant. Biol. 33, 417-424. VOGEL, H. J., and BONNER, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 128, 97-106. WERNER, R. (1968). Initiation and propagation of growing points in the DNA of phage T4. Cold Spring Harbor Symp. Quant. Biol. 33, 50-508. WIBERG, J. S. (1966). Mutants of bacteriophage T4 unable to cause breakdown of host DNA. Proc. Nat. Acad. Sci. U.S. 55, 614-621. Wu, R., and YEH, Y. C. (1970). DNA replication in mutants defective in gene 59 to T4 bacteriophage-infected E. coli. Bacterial. Proc., p. 167. WV, R., MA, F. J., and YEH, Y. C. (1972). Suppression of DNA-arrested synthesis in mutants defective in gene 59 of bacteriophage T4. Virology 47, 147-156. YAMAGUCHI, K., MURAKAMI, S., and YOSEIIKAWA, H. (1971). Chromosome-membrane association in Bacillus subtilis. I. DNA release from membrane fraction. Biochem. Biophys. Res. Commun. 44, 1559-1565.