Vol. 16, No. 2 Printed in U.S.A.

JOURNAL OF VIROLOGY, Aug. 1975, p. 412-419 Copyright ( 1975 American Society for Microbiology

Synthesis of Complex Forms of Bacteriophage iX174 Double-Stranded DNA in a Temperature-Sensitive dnaC Mutant of Escherichia coli C EVANGELIA G. KRANIAS1 AND LAWRENCE B. DUMAS* Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201

Received for publication 3 March 1975

Fast-sedimenting forms of bacteriophage kX174 double-stranded replicativeform DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicativeform replication. The data suggest that these higher-than-normal-molecularweight DNA molecules result from abnormal initiation of kX174 replicative-form DNA replication. Two to four percent of the intracellular double-stranded replicative-form (RF) of bacteriophage OX174 DNA are multiple-length molecules (1, 11, 15, 16). Most of these are multiplelength continuous circles, although interlocked monomer circles are also observed. Both kinds of structures are found to be the product of both DNA replication and DNA recombination. However, interlocked monomer circles are the predominant product of the DNA recombination pathway, and multiple-length continuous circles are the predominant product of the DNA replication pathway (1). Such complex forms of circular DNA are not unique to viruses. Multiple length forms of bacterial plasmid DNA have also been observed (7, 8, 9, 10). We have observed the accumulation of higherthan-normal-molecular-weight forms of OX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli at both the permissive and nonpermissive temperature for the dnaC protein activity. The dnaC protein is specifically -equired for the initiation of DNA synthesis (2, 17). These complex forms of OX174 RF DNA were also synthesized in the parental host strain, but not at the nonpermissive temperature in a dnaE mutant of the same parental host. The dnaE gene product, DNA polymerase III, is essential for DNA chain elongation (5, 14). We conclude that complex DNA molecules result from abnormal initiation of DNA synthesis. The conditions under which these molecules accumulate are reported, and Present address: Department of Biochemistry. Northwestern University Medical School, Chicago, Ill. 60611.

their relationship to the complex OX174 RF DNA molecules observed by others in normal infections is discussed. MATERIALS AND METHODS Bacteria and phage strains. LD301 and LD331 are, respectively, dnaEts, and dnaCts mutants of H502 (uvrA-, thyA-, endI-) previously described (4, 12). kX174 am3 (gene E) is a lysis-defective mutant. Infection and preparation of cell lysates. These procedures have been described (4). In these experiments phage infection was carried out in media supplemented with 0.01 lsg of thymine per ml. This low concentration of thymine was used to increase the specific activity of [3H ]thymidine-labeled intracellular OX174 DNA. In 0.01 jig of thymine per ml uninfected host cells double in number before multiplication ceases. The yield of kX174 am3 in this concentration of thymine is 15% of that in 2 ug of thymine per ml. The replication of OX174 am3 RF DNA in the dnaC mutant host is as temperaturesensitive in 0.01 ug of thymine per ml as in 2 gg per ml. Centrifugation analyses. The conditions of the zone sedimentation analyses in neutral pH sucrose have been described (12). Alkaline pH sucrose gradients consisted of 5 to 20% sucrose in 0.2 M NaOH, 0.8 M NaCl, 2 mM EDTA, and 0.1% Sarkosyl, pH 12.6. Cesium chloride density gradient analysis was carried out as described by Muller-Wecker et al. (13). The samples were spun in a Beckman type 65 rotor at 45,000 rpm for 48 h at 20 C. Electron microscopy. The intracellular phage DNA was extracted by using the procedure of Godson and Vapnek (6). The DNA was fractionated on neutral sucrose gradients, dialyzed against 50 mM Tris-hydrochloride, 1 mM EDTA, pH 8.1, at 4 C, and precipitated at -20 C by addition of 2 to 3

volumes of ethanol. 412

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SYNTHESIS OF COMPLEX OX174 DNA

The DNA samples were spread and shadowed by using the techniques described by Davis et al. (3). Chemicals. [Methyl-H lthymidine, 56 Ci/mmol, was purchased from Amersham/Searle. Mitomycin C, chloramphenicol, egg white lysozyme, and protease (type VI) were purchased from Sigma Chemical Co.

RESULTS Synthesis of abnormally high-molecularweight qX174 RF DNA. Previous experiments (12) showed that one of the major products of DNA synthesis in a 4X174-infected dnaCt8 mutant host at 41 C, during the doublestranded RF DNA replication stage, was a species that sedimented in a broad band at about the same rate as kX174 single-stranded DNA in high ionic strength medium (27S). We monitored the synthesis of this DNA under conditions where the specific activity of the radioactive label was increased, and examined its structure. To determine whether this DNA was singlestranded phage DNA or a high-molecular-weight form of 4X174 double-stranded DNA, liquid cultures of the dnaCts mutant LD331 were infected with OX174 am3 in the presence of 30 ,ug of chlorampenicol per ml. This concentration of chloramphenicol inhibits single-stranded synthesis, allowing prolonged RF replication (18). The phage DNA in these cells was labeled with [3H]thymidine at 30 and 41 C. 3H-labeled DNA that sedimented faster than the singlestranded phage DNA marker was seen in lysates of the infected cells pulse-labeled at 30 C (Fig. 1A), and pulse-labeled after shifting to 41 C (Fig. 1B). The normal products of OX174 RF DNA replication, RFI (21S) and RFII (16S), were also observed. The 3H-labeled fast-sedimenting DNA was also observed after longer pulses, and after chases at 30 and 41 C (see

below). The DNA in the fast-sedimenting bands from sucrose gradients was subjected to equilibrium buoyant density analysis (Fig. 2). Almost all of the DNA in this band from lysates of cells labeled at either temperature had the density of double-stranded DNA. The same was true of the fast-sedimenting DNA from lysates of cells that had been chased at both temperatures (data not shown). It was not an aggregate of normal OX174 RF DNA held together by proteins since it was stable to degradation by nonspecific proteases followed by heating 20 min at 56 C (Fig. 1). It was not host DNA since no radioactively labeled DNA sedimenting at the same rate in neutral pH sucrose gradients was found in extracts of uninfected host cells at 30 or 41 C in identical experiments (data not shown). We conclude therefore, that a higher-

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FIG. 1. Zone sedimentation of intracellular phage DNA from 4X174 am3-infected LD331. A liquid culture of bacteria was grown at 30 C to a cell density of 3 x 108 cells/ml on TPGA medium supplemented with 2 jug of thymine per ml. The cells were collected by centrifugation and suspended in 0.1 volume of TPG medium. Mitomycin C was added to 0.1 mg/ml. After 20 min in the dark the cells were collected and resuspended in 1 volume of TPGA medium supplemented with 0.01 Ag of thymine per ml. Phage (10 per cell) and chloramphenicol (30 ,g per ml) were added at zero time. After 55 min at 30 C 200 MCi of [3H]thymidine were added to 20 ml of the infected culture. Two minutes later this portion of the culture (A) was rapidly chilled. At 55 min after infection another 20-ml portion of the culture was shifted to 41 C. Fifteen minutes later 200 ,uCi of ['H]thymidine were added for 2 min, and the culture was rapidly chilled (B). The chilled cells were collected, washed, and lysed. The lysates were digested with protease, heated 20 min at 56 C, and sedimented through neutral pH sucrose gradients (16 h at 4 C at 25,000 rpm in a Beckman SW27 rotor). Thirty fractions were collected from the bottom of each gradient. The amount of radioactivity in 0.2 ml of each fraction was measured. Arrows indicate the positions of added marker "2P-labeled virus DNA.

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20 40 60 FRACTION NUMBER FIG. 2. Cesium chloride equilibrium buoyant density analysis of the fast-sedimenting OX174 DNA. The DNA in the fast-sedimenting bands from neutral pH sucrose gradients was dialyzed, concentrated by alcohol precipitation, and spun in CsCI gradients. 32P-labeled kX174 single-stranded DNA marker was added to each sample. Frame A represents the banding profile of the DNA from a lysate of kX174 am3-infected LD331 treated exactly as described for sample A in Fig. 1. Frame B represents the banding profile of the DNA from a lysate of infected cells labeled for 30 min at 41 C beginning at 5 min after the temperature shift-up. Frame C represents a

than-normal-molecular-weight form of OX174 double-stranded DNA was synthesized in this mutant host both at 30 and 41 C. The amount of high-molecular-weight kX174 DNA synthesized in LD331 during short pulses at 30 C was about the same in media containing 1 and 0.01 Ag of thymine per ml. This DNA represented 11 + 3 and 16 ± 3%, respectively, of the iX174 double-stranded DNA synthesized at these thymine concentrations. Similarly, this complex DNA was observed in OX174-infected LD331 that had not been treated with mitomycin C. It is not therefore an artifact of these experimental conditions. In experiments similar to those described in Fig. 1, we measured the amounts of the abnormally high-molecular-weight, OX174 doublestranded DNA synthesized at 30 and 41 C, and the rates of synthesis of the high-molecularweight and normal RF DNA at 41 C relative to those at 30 C. The data from three experiments is summarized in Table 1. These data show that the percentage of high-molecular-weight DNA synthesized during short pulses at 30 C was about the same in LD331 and the parent host strain H502. The rate of normal RF replication at 41 C in LD331 was about 70% less than at 30 C. The rate of synthesis of the high-molecular-weight OX174 DNA in both hosts at 41 C was at least as high as that at 30 C. These data suggest that the dnaC gene product is not TABLE 1. Synthesis of the high-molecular-weight and normal forms of qX174 RF DNA at 41 and 30 C in LD331 and H502a % HMWb

ExptA, LD331 B, LD331 C, H502

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a Cells were infected at 30 C in 30 of chloramphenicol per ml. Half of each culture was shifted to 41 C at 45 min after infection. Equal portions of each culture were pulse-labeled with [3H]thymidine for 2 min (LD331) and 5 min (H502) at 20 min (A) and (C) and 30 min (B) after the temperature shift. The rate of DNA synthesis was assumed to be proportional to the counts per minute incorporated during the pulse. bHMW, High molecular weight. c Total = HMW plus normal RF.

control where OX174 3H-labeled RF DNA was mixed with the 32P-labeled single-stranded DNA marker. Fractions were collected from the bottoms of the gradients. Arrows indicate the position of added 32P-labeled single-stranded OX1 74 DNA marker.

VOL. 16, 1975

essential for the synthesis of the high-molecular-weight form of /X174 DNA, although it is for the synthesis of normal /X174 RF DNA (12). Thus, at 41 C the synthesis of this complex DNA continues in the dnaC mutant host, while the synthesis of normal RF DNA is markedly inhibited. This results in an increase in the percentage of high-molecular-weight DNA at 41 C. Function of the complex forms of 4X174 DNA. We asked whether the complex forms of iX174 double-stranded DNA were intermediates in normal RF replication or dead-end products of abnormal DNA synthesis. The intracellular phage DNA in LD331 was pulselabeled at 41 C and chased at 30 and 41 C. Fast-sedimenting high-molecular-weight kX174 DNA was again detected in the lysate of the pulse-labeled culture (Fig. 3A). This complex DNA was not chased into normal RF DNA at either 30 (Fig. 3B) or 41 C (Fig. 3C). kX174 RF replication resumes at a near normal rate upon shifting down to 30 C under these conditions (unpublished observation). These data suggest that the complex form of kX174 DNA is not an intermediate in normal RF replication, but rather a dead-end product. Synthesis of the complex form of OX174 DNA in a dnaE mutant. In the temperaturesensitive dnaE mutant LD301 fast-sedimenting kX174 DNA was synthesized at 30 C but not at 41 C (Fig. 4). This suggests that DNA polymerase III-catalyzed chain elongation is necessary for the synthesis of the high-molecular-weight qX174 DNA. Structure of the complex form of OX174 DNA. All of our data relevant to the structure of complex form of kX174 DNA suggest that it consists of the kinds of oligomeric forms of OX174 monomeric RF DNA observed by others in infections of normal host cells (1, 11, 15, 16). When this DNA from sucrose gradients was dialyzed, concentrated by alcohol precipitation, and resedimented, we observed a partial conversion to slower-sedimenting species. About one-third still sedimented at the original rate (approximately 30S), while the remainder sedimented at 14 to 30S (data not shown). Previous studies showed that supercoiled circular dimers of OX174 RF DNA sediment at 29S, relaxed circular dimers at 21S, and higher order relaxed FIG. 3. Zone sedimentation of phage DNA extracted from 4,X174 am3-infected LD331 pulse-labeled and chased during RF replication. The conditions for culturing the cells, mitomycin C treatment, and infection were identical to those described in

SYNTHESIS OF COMPLEX OX174 DNA

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Fig. 1. At 40 min after infection 10-ml portions of the culture were shifted to 41 C. At 20 min after the shift 100 ACi of [(H]thymidine were added to each. Five minutes later one portion was rapidly chilled (A), while the other two were filtered. The latter two were then washed with 10 ml of medium containing 200 jAg of thymine, 2 mg of thymidine, and 30 lAg of chloramphenicol per ml. The cells were resuspended in 10 ml of the same medium and incubated 30 min at 30 (B) and 41 C (C). The cells were collected, washed, lysed, digested with protease, heated to 56 C for 20 min, and sedirrtented as described in Fig. 1. Arrows indicate the positions of added marker 32P-labeled 4X174 single-stranded DNA.

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in alkaline pH sucrose gradients showed three major bands (Fig. 5). The DNA in the middle band sedimented at the rate of denatured monomeric RFI DNA. The DNA in the band closest to the bottom of the gradient sedimented at the rate expected of denatured oligomeric RFI DNA. The DNA in the slowest-sedimenting band consisted of denatured single strands derived from relaxed double-stranded DNA molecules. When sedimented for longer times (data not shown), the band was shown to be more heterogeneous than that of an added single-stranded circular DNA marker. The data suggested the presence of monomeric circular and linear DNA strands, as well as oligomeric single-strands. When other preparations of DNA from neutral pH sucrose gradients were spun to equilibrium in CsCl gradients containing ethidium bromide (100 ,ug/ml), about 20% banded at the position of supercoiled DNA. When dialyzed, concentrated DNA from neutral pH sucrose gradients was spread and exam-

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FIG. 4. Zone sedimentation of phage DNA extracted from OX1 74 am3-infected LD301 pulse-labeled during RF replication. The conditions were identical to those described in Fig. 1. After the mitomycin C treatment the cells were suspended in I volume of TPGA medium supplemented with 0.01 ,ug of thymine per ml. Phage were added at a multiplicity of 10 and chloramphenicol at 30 gg per ml. After 45 min at 30 C half of the culture was shifted to 41 C. At 20 and 45 min after the shift 10-ml portions of the 30 and 41 C cultures were pulselabeled for 2 min with [3H]thymidine, 10 uCi per ml. A, 20 min, 30 C; B, 20 min, 41 C; C, 45 min, 30C;D,45min,41 C.

oligomers somewhat faster than 21S (11). Supercoiled and relaxed monomeric RF DNA molecules sediment at 21 and 16S, respectively. Thus, before these manipulations the complex form of OX174 DNA observed in the dnaC mutant host sedimented at a rate expected of supercoiled dimeric and higher oligomeric forms of RF DNA. After these manipulations a heterogeneous population of species sedimenting at the rates expected of relaxed forms and breakdown products of these complex DNA molecules was observed. Zone sedimentation of the dialyzed, concentrated DNA from neutral pH sucrose gradients

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5. Zone sedimentation of the high-molecularkX174 DNA in an alkaline sucrose gradient. was treated as described for sample B in At 5 min after the shift to 41 C 200 MCi of ['H]thymidine were added. Thirty minutes later the infected cells were collected and treated as described in Fig. 1. The DNA from the fast-sedimenting band was dialyzed and concentrated by alcohol precipitation. This DNA sample was spun in an alkaline pH sucrose gradient for 3.5 h at 38,000 rpm in a Beckman SW40 rotor at 5 C. Total radioactivity was measured in each fraction collected from the bottom of the

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VOL. 16, 1975

SYNTHESIS OF COMPLEX OX174 DNA

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KRANIAS AND DUMAS

ined by electron microscopy, monomer and complex forms of OX174 DNA were observed. The complex forms included tetramers, trimers, and dimers, some continuous, some apparently catenated. Some examples are shown in Fig. 6. These data suggest no unique structure for the complex qX174 DNA observed in the dnaC mutant host. The data indicate that these complex DNA molecules are similar in structure to at least some of those found by other investigators in OX174 infections of normal cells.

DISCUSSION High-molecular-weight double-stranded forms of OX174 DNA were synthesized during the period of RF replication (stage II) at both 30 and 41 C in a temperature-sensitive dnaC mutant host. dnaC mutants are defective in the initiation of DNA synthesis (2, 17). At 41 C the rate of OX174 RF DNA synthesis in this mutant host was 30% of that at 30 C (Table 1). The highmolecular-weight DNA was synthesized at least as fast at 41 C as at 30 C. This suggests that the formation of these complex DNA structures is not dependent upon the activity of the dnaC initiation protein. Our data suggest that this complex DNA is the product of DNA synthesis, rather than DNA recombination. Its formation required the activity of the dnaE protein, DNA polymerase III (Fig. 4). This enzyme is essential for normal OX174 RF replication (4). Also, more radioactively labeled high-molecular-weight OX174 DNA was made at 41 C during a short pulse than at 30 C, while less radioactively labeled RF DNA was made (Fig. 1, Table 1). If the radioactivity labeled complex DNA were a product of recombination between normal RF DNA molecules synthesized during the period of the pulse, the rate of its formation should decrease at 41 C. The high-molecular-weight 4X174 DNA was not chased into kX174 RF I and RF II DNA molecules, the products of normal RF replication, at 30 or 41 C (Fig. 3). This indicates that this complex DNA is a dead-end product of abnormal DNA synthesis, not an intermediate in normal RF replication. High-molecular-weight forms of OX174 double-stranded DNA have been observed previously in normal infections, and in infections where RF replication was inhibited by phage mutations and drugs (1, 11, 15, 16). Both catenated and continuous circles were observed. These structures apparently can arise from both DNA replication and DNA recombination. Closed circular complex DNA structures repre-

J. VIROL.

sent 2 to 4% of the supercoiled RF DNA

synthesized under these conditions (11). Those complex DNA molecules stable to repeated centrifugation represent about 4% of the total double-stranded RF DNA in the cell (1). In our experiments the complex DNA molecules represented approximately 15% of the total OX174 RF DNA synthesized at 30 C. Twenty to fifty percent of this complex DNA was completely closed circular, as seen by analysis in alkaline pH sucrose gradients and in CsCl-ethidium bromide gradients. About onethird still sedimented at 30S at neutral pH after the initial purification on sucrose gradients. The levels of complex kX174 DNA that we observed in infections at 30 C are therefore comparable to those observed in previous investigations, the difference being that we did not select only the completely closed circular DNA fraction or only the fraction stable to repeated centrifugation. Thus the kinds of DNA molecules that accumulate in the dnaC mutant host at the nonpermissive temperature have also been observed in this host at the permissive temperature and in normal host cells. These high-molecular-weight OX174 DNA molecules apparently result from abnormal initiation of RF DNA synthesis since they continue to be synthesized at a normal rate under conditions where the initiation of normal RF replication is inhibited. The abnormal initiation pathway is used infrequently in normal infections since only a small amount of this complex DNA is synthesized. This pathway predominates only when the normal one is inhibited, leading to an accumulation of complex qX174 DNA. ACKNOWLEDGMENTS These results are drawn from a thesis submitted by E. G. K. to Northwestern University in partial fulfillment of the requirement for the Ph.D. degree. We thank R. Ryan for his help with the electron microscopy, and H. Swift for the use of his electron microscope facilities. This work was supported by a Public Health Service research grant (AI-9882) and research career development award (AI-70, 632) to L. B. D. from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED 1. Benbow, R. M., M. Eisenberg, and R. L. Sinsheimer. 1972. Multiple length DNA molecules of bacteriophage OX174. Nature (London) New Biol. 237:141-144. 2. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 3. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, p. 413-428. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. XXI. Academic Press Inc., New York.

VOL. 16, 1975 4. Dumas, L. B., and C. A. Miller. 1973. Replication of bacteriophage 4X174 DNA in a temperature-sensitive dnaE mutant of Eseherichia coli C. J. Virol. 11:848-855. 5. Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler, and C. Barnoux. 1971. Analysis of DNA polymerases II and m in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 68:3150-3153. 6. Godson, G. N., and D. Vapnek. 1973. A simple method of preparing large amounts of OX174 RF supercoiled DNA. Biochim. Biophys. Acta 299:516-520. 7. Goebel, W. 1970. Studies on extrachromosomal DNA. Replication of the colicinogenic factor col El in two temperature-sensitive mutants of Escherichia coli defective in DNA replication. Eur. J. Biochem. 15:311-320. 8. Goebel, W. 1974. Studies on the initiation of plasmid DNA replication. Eur. J. Biochem. 41:51-62. 9. Goebel, W., and D. R. Helinski. 1968. Generation of higher multiple circular DNA forms in bacteria. Proc. Natl. Acad. Sci. U.S.A. 61:1406-1413. 10. Goebel, W., and J. Kraft. 1974. Complex col El DNA in Escherichia coli and Proteus mirabilis. Mol. Gen. Genet. 129:149-166. 11. Gordon, C. M., M. G. Rush, and A. C. Warner. 1970. Complex replicative form molecules of bacteriophage *X174 and S13 su 105. J. Mol. Biol. 47:495-503.

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12. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophage OX174 DNA in a temperature-sensitive dnaC mutant of Escherichia coli C. J. Virol. 13: 146-154. 13. Muller-Wecker, H., K. Geider, and H. Hoffmann-Berling. 1972. DNA synthesis in nucleotide-permeable Escherichia coli cells. IV. Mode of 1X174 replicative form DNA synthesis and the template involved. J. Mol. Biol. 69:319-331. 14. Nusslein, V., B. Otto, F. Bonhoeffer, and H. Schaller. 1971. Function of DNA polymerase III in DNA replication. Nature (London) 234:285-286. 15. Rush, M. G., and R. C. Warner. 1968. Multiple length rings of OX174 and S13 replicative forms. III. A possible intermediate in recombination. J. Biol. Chem. 243: 4821-4826. 16. Rush, M. G., A. K. Kleinschmidt, W. Hellman, and R. C. Warner. 1967. Multiple length rings in preparation of 4X174 replicative form. Proc. Natl. Acad. Sci. U.S.A. 58:1676-1683. 17. Schulbach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escherichia coli. J. Mol. Biol. 74:205-221. 18. Sinsheimer, R. L., B. Starman, C. Nagler, and S. Guthrie. 1962. The process of infection with bacteriophage OX174. I. Evidence for a replicative form. J. Mol. Biol. 4:142-160.

Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonp...
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