Vol. 16, No. 3 Printed in U.S.A.
JOURNAL OF VIROLOGY, Sept. 1975, p. 575-580 Copyright 0 1975 American Society for Microbiology
Rifampin Inhibition of Bacteriophage OX174 Parental Replicative-Form DNA Synthesis in an Escherichia coli dnaC Mutant LAWRENCE B. DUMAS,* CHRISTINE A. MILLER, AND MARVIN L. BAYNE
Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201 Received for publication 24 February 1975
The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage 4X174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, OX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in OX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of OX174 DNA are discussed.
In the first stage of the replication of bacteriophage OX174 DNA in its host, Escherichia coli, a complementary DNA strand is synthesized on the infecting single-stranded DNA template (parental replicative-form [RF] synthesis). This stage is catalyzed by preexisting host cell enzymes; i.e., it requires no phage-induced protein synthesis (11). The initiation of the synthesis of the complementary DNA strand requires RNA synthesis (8) and is resistant to rifampin (8, 10), a specific inhibitor of the host cell DNAdependent RNA polymerase (12, 15). In cell-free extracts of E. coli, the rifampinresistant synthesis of the complementary OX174 DNA strand requires the participation of the protein product of the host cell dnaC,gene (7, 16). DNA-dependent RNA polymerase-catalyzed synthesis of an RNA primer can obviate the requirement for the dnaC protein (13, 16). The E. coli dnaC protein is required for the initiation of cycles of chromosome replication in vivo (1, 9), as well as for the replication of the double-stranded replicative form of 4X174 DNA (5). However, OX174 parental RF synthesis occurs with near-normal efficiency in a dnaC-defective mutant of E. coli, suggesting that the dnaC protein is not absolutely required for the initation of this synthesis in vivo (5). Since the synthesis of the OX174 complementary DNA strand in vitro requires the dnaC protein only when DNA-dependent RNA polymerase activity is inhibited, we asked whether the same might be true in the cell. We present evidence here showing that the host cell dnaC protein is indeed required for the synthesis of the OX174 complementary DNA strand in vivo 575
when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase. MATERIALS AND METHODS Bacteria and phage. The mutant host strain LD332 is a nitrosoguanidine-induced temperaturesensitive mutant of H502 (uvrA -, thyA -, endIl) isolated in our laboratory. The temperature-sensitive locus lies within the region of the chromosome carried by F-prime factor 101 and cotransduces with dra-1 at a frequency of 0.29. These data indicate that this marker maps at the dnaC locus (14). This temperature-sensitive mutant has the same properties as the 4X174-sensitive dnaC mutant previously described (5), except that it is less leaky for DNA synthesis at the nonpermissive temperature. Host strain LD3321 is a spontaneous temperatureinsensitive revertant of LD332. Host strain LD3322 is a spontaneous rifampin-resistant mutant of LD332. 4OX174 am3 is a lysis-defective gene E mutant. Infection and lysis. A 100-ml culture of host bacteria was grown on TPGA medium (3) supplemented with 2 tsg of thymine per ml at 30 C to a cell density of 5 x 10' cells/ml. The cells were collected by centrifugation, resuspended in starvation buffer (2), and treated with 100 ,g of mitomycin C per ml for 20 min. The cells were again collected by centrifugation and resuspended in 100 ml of TPGA medium supplemented with 1 ;g of thymine per ml. Twenty-five-milliliter portions were incubated at 30 and 41 C with aeration for 30 min. Rifampin was added to 200 jg/ml to one of the cultures at 25 min after resuspension. At the end of the 30-min preincubation (zero time), 32P-labeled OX174 am3 was added to each culture of LD332 and LD3321 at a multiplicity of infection of 0.2, and to each culture of LD3322 at a multiplicity of infection of 3. The specific activity of the phage was 10-' counts/min per PFU. [JH]Thymidine (100 MCi) was added to each culture at the same time. After 20
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min the cultures were rapidly chilled. The cells were collected by centrifugation, washed twice in 50 mM sodium tetraborate, 6 mM EDTA, pH 9, and once in 50 mM Tris-hydrochloride, 3 mM EDTA, pH 8, and resuspended in 1 ml of 50 mM Tris-hydrochloride, 10 mM EDTA, 0.2 mg of lysozyme, pH 8. After 20 min of incubation at 0 C, Sarkosyl was added to 3%. This lysate was incubated for 16 h at 0 C. Protease (0.5 mg/ml, type VI, Sigma Chemical Co.) was then added, and the suspension was incubated for 6 h at 37 C. Finally, the mixture was heated for 20 min at b6 C to further disrupt DNA-protein aggregates. Zone sedimentation analysis. Each lysate was layered onto a 36-ml linear gradient of 5 to 20% sucrose in 50 mM potassium phosphate, 2 mM EDTA, 1 M NaCl, pH 7. The gradients were spun for 16 h (17 h in the LD 3322 experiment) at 15 C at 24,000 rpm in a Beckman SW27 rotor. Fractions (0.6 ml) were collected from the bottoms of the tubes into 1-dram shell vials. Water (0.4 ml) and scintillation fluid (3 ml of 4 g of 2,5-diphenyloxazole per liter in 2 volumes of toluene to 1 volume of Triton X-100) were added, and the radioactivity in each sample was measured. RESULTS
We monitored the conversion of infecting "2P-labeled 4X174 virus DNA to doublestranded parental RF DNA at 30 and 41 C in the temperature-sensitive mutant host LD332 (uvrA-, thyA-, endI-, dnaCt) in the presence and absence of rifampin. Lysates of infected cells were layered onto sucrose gradients containing 1 M NaCl. On such gradients the double-stranded, supercoiled RFI DNA (21S) and the double-stranded, relaxed RFII DNA (16S) can be separated from the single-stranded parent phage DNA (27S). The efficiency of parental RF synthesis was determined by measuring the amount of "2P label in the RF DNA bands relative to that in the control (30 C, no rifampin). If the host cell dnaC protein were required for the initiation of OX174 parental RF DNA synthesis when the DNA-dependent RNA polymerase activity was inhibited, but not when it was fully active, we would expect a near-normal efficiency of parental RF synthesis at 41 C without rifampin and at 30 C in rifampin, but a marked reduction in efficiency at 41 C in rifampin.
4X174 parental RF DNA was synthesized at 41 C (Fig. 1B). The amount of 8H label incorporated into RF DNA was only slightly above background since replication of the doublestranded RF DNA was inhibited at this temperature (5). The amount of 32P label in RF DNA at 41 C was 45% of that at 30 C. In the temperature-insensitive revertant, the amount of 32p label in RF DNA at 41 C was 70% of that at 30 C (compare Fig. 2B with 2A). In the rifampin-
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resistant derivative of the dnaC mutant host, the amount of 3"P label found in RF DNA was 105% of that at 30 C (compare Fig. 3B with 3A). These results are consistent with the previous observation that OX174 parental RF synthesis occurs under conditions where the host dnaC protein is inactive (5). 4X174 parental RF DNA was also synthesized at 30 C in the presence of 200 ,ug of rifampin per ml (Fig. 1C; ref. 8, 10). The amount of 3"P label found in RF DNA molecules was 80% of that at 30 C without rifampin. The fact that more 3H label was found in RF DNA under these conditions than at 41 C (Fig. 1B) probably means that the RF DNA replicated slowly at 30 C in rifampin. In the temperature-insensitive revertant host strain, the amount of "2P label in RF DNA at 30 C in rifampin was also 80% of that at 30 C without rifampin (compare Fig. 2C with 2A). In the rifampin-resistant derivative of the dnaC mutant host, the amount of "P label in RF DNA at 30 C in rifampin was 130% of that at 30 C without rifampin (compare Fig. 3C with 3A). The amount of 32P label found in RF DNA at 41 C in rifampin in the dnaC mutant host (Fig. 1D) was only 10% of that in the 30 C control without rifampin, whereas in the temperatureinsensitve revertant (Fig. 2D) and the rifampinresistant derivative (Fig. 3D) there was 100% as much as at 30 C without rifampin. Thus, the efficiency of OX174 parental RF DNA synthesis was markedly reduced at 41 C in rifampin in the dnaC mutant, but not in the control strains. The efficiency was near normal in the dnaC mutant at 41 C without rifampin and at 30 C in rifampin. These data indicate that the rifampin sensitivity of 4X174 parental RF DNA synthesis at 41 C in the dnaC mutant host was due to the inhibition of the DNA-dependent RNA polymerase activity, and that the temperature sensitivity in the presence of rifampin was due to the temperature sensitivity of the dnaC gene product. When either the dnaC protein activity or
the DNA-dependent RNA polymerase activity alone was inhibited, qX174 parental RF DNA synthesis occurred at near-normal efficiency. However, when the dnaC protein activity and the DNA-dependent RNA polymerase activity were simultantously inhibited, the synthesis of 4X174 parental RF DNA was 90% inhibited. In addition, we have observed that the parental RF DNA synthesized at 30 C in LD332 in the presence and absence of rifampin is stable to further incubation at 41 C (data not shown). This observation negates the possibility that parental RF DNA might have been synthesized
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FRACTION NUMBER FIG. 1. Zone sedimentation of intracellular phage DNA extracted from 0X1 74 am3-infected LD332 (dnaCt8, rifampin sensitive). The experimental details are outlined in the text. (A) Incubated and infected at 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin. The three major 32P-labeled bands in each gradient correspond to 27S single-stranded phage DNA, 21S supercoiled double-stranded RFI DNA, and 16S relaxed double-stranded RFII DNA. Sedimentation was from right to left. Unincorporated ['H]thymidine was found at the top of each gradient.
at 41 C in rifampin under the conditions described in Fig. 1 and then degraded or chased into a faster-sedimenting species. The small amount of 3H label found at the position of 3"P-labeled single-stranded DNA in Fig. 1B, C, and D has not been further examined. In other experiments where there was considerably less "2P label at this position, no 'H label was detected. In the experiment presented here, it was present in about the same amounts when parental RF synthesis was normal and when it was 90% inhibited, as well as in the absence of rifampin and in its presence, where protein synthesis was inhibited. Since RF DNA serves as the template for single-stranded virus DNA synthesis and since newly synthesized proteins are also essential for singlestranded DNA synthesis, this 'H label probably does not represent newly synthesized singlestranded DNA. We cannot exclude the possibil-
ity that it represents short segments of complementary-strand DNA on the infecting viralstrand templates.
DISCUSSION These data show that the host dnaC protein is required for OX174 parental RF DNA synthesis, presumably in the initiation of the synthesis of the complementary DNA strand, when rifampin is added at a concentration that inhibits the DNA-dependent RNA polymerase activity of the host. It is not required when rifampin is absent. In the rifampin-resistant host, whose DNA-dependent RNA polymerase is presumably resistant to rifampin, the dnaC protein is not required when rifampin is added. We conclude therefore that the DNA-dependent RNA polymerase activity of the host cell obviates the need for the dnaC protein. DNA-dependent
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FRACTION NUMBER FIG. 2. Zone sedimentation of intracellular phage DNA from OX1 74 am3-infected LD3321 (dnaC+, rifampin sensitive). (A) 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin.
RNA polymerase catalyzes the synthesis of RNA chains on DNA templates. This RNA synthesis might obviate the need for the dnaC protein by directly providing a primer for DNA synthesis. Alternatively, the RNA may play a structural role in the initiation of DNA synthesis, thereby obviating the need for the dnaC protein. Finally, the DNA-dependent RNA polymerase may function not by synthesizing an RNA molecule, but as a structural component in an initiation complex. It seems clear that the RNA polymerase is not needed for mRNA synthesis since no new protein synthesis is required for OX174 parental RF synthesis (11). We cannot conclude that RNA polymerase and the dnaC protein catalyze the same reaction in the initiation of DNA synthesis, and that one protein can substitute directly for the other. RNA polymerase certainly cannot substitute for the dnaC protein in the semiconservative replication of the double-stranded replicative form of OX174 DNA (Fig. 1B; ref. 5), the second stage in kX174 DNA synthesis. The specificities of the two proteins must therefore be different. The same final products appear to be made no matter which of the two proteins is used in
the initiation of OX174 parental RF DNA synthesis. The OX174 RFI DNA molecules made at 30 C in the presence of rifampin and those made at 41 C in the absence of rifampin are supercoiled and stable to alkali, indicating that the DNA chains are covalently closed and free of ribose-containing nucleotides that might have been used in initiation by RNA polymerase as well as by the dnaC protein (data not shown). However, initiation might occur at a different site by a different mechanism in each case. Although DNA-dependent RNA polymerase can obviate the requirement for the dnaC protein in OX174 parental RF synthesis in vivo, the host dnaB and dnaG proteins are absolutely required (3, 6). The in vivo role of DNA-dependent RNA polymerase in this DNA synthesis is thus not simply a catalysis of random RNA primers that are elongated by DNA polymerase III assisted by the DNA unwinding protein, a pathway that is effective in vitro (4, 13, 18). Instead, the data suggest that DNA-dependent RNA polymerase substitutes only for the dnaC protein in the initiation of DNA synthesis, and that the dnaB and dnaG proteins are essential for the synthesis of the product DNA strand.
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FRACTION NUMBER FIG. 3. Zone sedimentation of intracellular phage DNA from 4X174 am3-infected LD332 (dnaCt8, rifampin resistant). (A) 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin.
Indeed, the two different initiation proteins probably participate in the same 4X174-specific parental RF DNA synthesis pathway now reconstituted in vitro in which the host dnaB, dnaG, and dnaE proteins play essential roles (7, 17). The participation of DNA-dependent RNA polymerase in the initiation of this OX174-specific parental RF DNA synthesis pathway in vitro has not yet been demonstrated. In fact, this enzyme can be specifically inhibited with respect to its ability to synthesize random RNA primers in the initiation of XX174 DNA synthesis in vitro. This inhibition requires the combined action of the DNA unwinding protein and a small protein that associates with the RNA polymerase (4, 19). However, our data suggest that in the cell there is sufficient DNA-dependent RNA polymerase activity to catalyze a more specific initiation of kX174 parental RF DNA synthesis. ACKNOWLEDGMENTS 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. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 2. Denhardt, D. T., and R. L. Sinsheimer. 1965. The process of infection with bacteriophage 4X174. III. Phage maturation and lysis after synchronized infection. J. Mol. Biol. 12:641-646. 3. Dumas, L. B., and C. A. Miller. 1974. Inhibition of bacteriophage 4X174 DNA replication in dnaB mutants of Escherichia coli C. J. Virol. 14:1369-1379. 4. Hurwitz, J., S. Wickner, and M. Wright. 1973. Studies on in vitro DNA synthesis. II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E. coli. Biochem. Biophys. Res. Commun. 51:257-267. 5. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophage 4X174 DNA in a temperature-sensitive dnaC mutant of Escherichia coli C. J. Virol. 13: 146-154. 6. McFadden, G., and D. T. Denhardt. 1974. Mechanism of replication of OX174 single-stranded DNA. IX. Requirement for the Escherichia coli drnG protein. J. Virol. 14:1070-1075. 7. Schekman, R., A. Weiner, and A. Kornberg. 1974. Multienzyme systems of DNA replication. Science 186:987-993.
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8. Schekman, R., W. Wickner, 0. Westergaard, D. Brutlag, K. Geider, L. L. Bertsch, and A. Kornberg. 1972. Initiation of DNA synthesis: synthesis of OX174 replicative form requires RNA synthesis resistant to rifampicin. Proc. Natl. Acad. Sci. U.S.A. 69:26912695. 9. Schubach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escherichia coli. J. Mol. Biol. 74:205-221. 10. Silverstein, S., and D. Billen. 1971. Transcription: role in the initiation and replication of DNA synthesis in Escherichia coli and OX174. Biochim. Biophys. Acta 247:383-390. 11. Sinsheimer, R. L., R. Knippers, and T. Komano. 1968. Stages in the replication of bacteriophage OX174 in vivo. Cold Spring Harbor Symp. Quant. Biol. 33: 443-447. 12. Sippel, A., and G. Hartmann. 1968. Mode of action of rifampicin on the RNA polymerase reaction. Biochim. Acta 157:218-219. 13. Tobak, H. F., J. Griffith, K. Geider, H. Schaller, and A.
14.
15.
16.
17.
18.
Kornberg. 1974. Initiation of deoxyribonucleic acid synthesis. VII. A unique location of the gap in the M13 replicative duplex synthesized in vitro. J. Biol. Chem. 249:3049-3054. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutants temperature sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. Wehrli, W., J. Nuesch, F. Knusel, and M. Staehelin. 1968. Action of rifampicins on RNA polymerase. Biochim. Biophys. Acta 157:215-217. Wickner, R. B., M. Wright, S. Wickner, and J. Hurwitz. 1972. Conversion of OX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:3233-3237. Wickner, S., and J. Hurwitz. 1974. Conversion of OX174 viral DNA to double-stranded form by purified Escherichia coli proteins. Proc. Natl. Acad. Sci. U.S.A. 71:4120-4124. Wickner, W., and A. Kornberg. 1974. A novel form of RNA polymerase from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:4425-4428.
JOURNAL OF VIROLOGY, Sept. 1975, p. 575-580 Copyright 0 1975 American Society for Microbiology
Rifampin Inhibition of Bacteriophage OX174 Parental Replicative-Form DNA Synthesis in an Escherichia coli dnaC Mutant LAWRENCE B. DUMAS,* CHRISTINE A. MILLER, AND MARVIN L. BAYNE
Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201 Received for publication 24 February 1975
The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage 4X174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, OX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in OX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of OX174 DNA are discussed.
In the first stage of the replication of bacteriophage OX174 DNA in its host, Escherichia coli, a complementary DNA strand is synthesized on the infecting single-stranded DNA template (parental replicative-form [RF] synthesis). This stage is catalyzed by preexisting host cell enzymes; i.e., it requires no phage-induced protein synthesis (11). The initiation of the synthesis of the complementary DNA strand requires RNA synthesis (8) and is resistant to rifampin (8, 10), a specific inhibitor of the host cell DNAdependent RNA polymerase (12, 15). In cell-free extracts of E. coli, the rifampinresistant synthesis of the complementary OX174 DNA strand requires the participation of the protein product of the host cell dnaC,gene (7, 16). DNA-dependent RNA polymerase-catalyzed synthesis of an RNA primer can obviate the requirement for the dnaC protein (13, 16). The E. coli dnaC protein is required for the initiation of cycles of chromosome replication in vivo (1, 9), as well as for the replication of the double-stranded replicative form of 4X174 DNA (5). However, OX174 parental RF synthesis occurs with near-normal efficiency in a dnaC-defective mutant of E. coli, suggesting that the dnaC protein is not absolutely required for the initation of this synthesis in vivo (5). Since the synthesis of the OX174 complementary DNA strand in vitro requires the dnaC protein only when DNA-dependent RNA polymerase activity is inhibited, we asked whether the same might be true in the cell. We present evidence here showing that the host cell dnaC protein is indeed required for the synthesis of the OX174 complementary DNA strand in vivo 575
when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase. MATERIALS AND METHODS Bacteria and phage. The mutant host strain LD332 is a nitrosoguanidine-induced temperaturesensitive mutant of H502 (uvrA -, thyA -, endIl) isolated in our laboratory. The temperature-sensitive locus lies within the region of the chromosome carried by F-prime factor 101 and cotransduces with dra-1 at a frequency of 0.29. These data indicate that this marker maps at the dnaC locus (14). This temperature-sensitive mutant has the same properties as the 4X174-sensitive dnaC mutant previously described (5), except that it is less leaky for DNA synthesis at the nonpermissive temperature. Host strain LD3321 is a spontaneous temperatureinsensitive revertant of LD332. Host strain LD3322 is a spontaneous rifampin-resistant mutant of LD332. 4OX174 am3 is a lysis-defective gene E mutant. Infection and lysis. A 100-ml culture of host bacteria was grown on TPGA medium (3) supplemented with 2 tsg of thymine per ml at 30 C to a cell density of 5 x 10' cells/ml. The cells were collected by centrifugation, resuspended in starvation buffer (2), and treated with 100 ,g of mitomycin C per ml for 20 min. The cells were again collected by centrifugation and resuspended in 100 ml of TPGA medium supplemented with 1 ;g of thymine per ml. Twenty-five-milliliter portions were incubated at 30 and 41 C with aeration for 30 min. Rifampin was added to 200 jg/ml to one of the cultures at 25 min after resuspension. At the end of the 30-min preincubation (zero time), 32P-labeled OX174 am3 was added to each culture of LD332 and LD3321 at a multiplicity of infection of 0.2, and to each culture of LD3322 at a multiplicity of infection of 3. The specific activity of the phage was 10-' counts/min per PFU. [JH]Thymidine (100 MCi) was added to each culture at the same time. After 20
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min the cultures were rapidly chilled. The cells were collected by centrifugation, washed twice in 50 mM sodium tetraborate, 6 mM EDTA, pH 9, and once in 50 mM Tris-hydrochloride, 3 mM EDTA, pH 8, and resuspended in 1 ml of 50 mM Tris-hydrochloride, 10 mM EDTA, 0.2 mg of lysozyme, pH 8. After 20 min of incubation at 0 C, Sarkosyl was added to 3%. This lysate was incubated for 16 h at 0 C. Protease (0.5 mg/ml, type VI, Sigma Chemical Co.) was then added, and the suspension was incubated for 6 h at 37 C. Finally, the mixture was heated for 20 min at b6 C to further disrupt DNA-protein aggregates. Zone sedimentation analysis. Each lysate was layered onto a 36-ml linear gradient of 5 to 20% sucrose in 50 mM potassium phosphate, 2 mM EDTA, 1 M NaCl, pH 7. The gradients were spun for 16 h (17 h in the LD 3322 experiment) at 15 C at 24,000 rpm in a Beckman SW27 rotor. Fractions (0.6 ml) were collected from the bottoms of the tubes into 1-dram shell vials. Water (0.4 ml) and scintillation fluid (3 ml of 4 g of 2,5-diphenyloxazole per liter in 2 volumes of toluene to 1 volume of Triton X-100) were added, and the radioactivity in each sample was measured. RESULTS
We monitored the conversion of infecting "2P-labeled 4X174 virus DNA to doublestranded parental RF DNA at 30 and 41 C in the temperature-sensitive mutant host LD332 (uvrA-, thyA-, endI-, dnaCt) in the presence and absence of rifampin. Lysates of infected cells were layered onto sucrose gradients containing 1 M NaCl. On such gradients the double-stranded, supercoiled RFI DNA (21S) and the double-stranded, relaxed RFII DNA (16S) can be separated from the single-stranded parent phage DNA (27S). The efficiency of parental RF synthesis was determined by measuring the amount of "2P label in the RF DNA bands relative to that in the control (30 C, no rifampin). If the host cell dnaC protein were required for the initiation of OX174 parental RF DNA synthesis when the DNA-dependent RNA polymerase activity was inhibited, but not when it was fully active, we would expect a near-normal efficiency of parental RF synthesis at 41 C without rifampin and at 30 C in rifampin, but a marked reduction in efficiency at 41 C in rifampin.
4X174 parental RF DNA was synthesized at 41 C (Fig. 1B). The amount of 8H label incorporated into RF DNA was only slightly above background since replication of the doublestranded RF DNA was inhibited at this temperature (5). The amount of 32P label in RF DNA at 41 C was 45% of that at 30 C. In the temperature-insensitive revertant, the amount of 32p label in RF DNA at 41 C was 70% of that at 30 C (compare Fig. 2B with 2A). In the rifampin-
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resistant derivative of the dnaC mutant host, the amount of 3"P label found in RF DNA was 105% of that at 30 C (compare Fig. 3B with 3A). These results are consistent with the previous observation that OX174 parental RF synthesis occurs under conditions where the host dnaC protein is inactive (5). 4X174 parental RF DNA was also synthesized at 30 C in the presence of 200 ,ug of rifampin per ml (Fig. 1C; ref. 8, 10). The amount of 3"P label found in RF DNA molecules was 80% of that at 30 C without rifampin. The fact that more 3H label was found in RF DNA under these conditions than at 41 C (Fig. 1B) probably means that the RF DNA replicated slowly at 30 C in rifampin. In the temperature-insensitive revertant host strain, the amount of "2P label in RF DNA at 30 C in rifampin was also 80% of that at 30 C without rifampin (compare Fig. 2C with 2A). In the rifampin-resistant derivative of the dnaC mutant host, the amount of "P label in RF DNA at 30 C in rifampin was 130% of that at 30 C without rifampin (compare Fig. 3C with 3A). The amount of 32P label found in RF DNA at 41 C in rifampin in the dnaC mutant host (Fig. 1D) was only 10% of that in the 30 C control without rifampin, whereas in the temperatureinsensitve revertant (Fig. 2D) and the rifampinresistant derivative (Fig. 3D) there was 100% as much as at 30 C without rifampin. Thus, the efficiency of OX174 parental RF DNA synthesis was markedly reduced at 41 C in rifampin in the dnaC mutant, but not in the control strains. The efficiency was near normal in the dnaC mutant at 41 C without rifampin and at 30 C in rifampin. These data indicate that the rifampin sensitivity of 4X174 parental RF DNA synthesis at 41 C in the dnaC mutant host was due to the inhibition of the DNA-dependent RNA polymerase activity, and that the temperature sensitivity in the presence of rifampin was due to the temperature sensitivity of the dnaC gene product. When either the dnaC protein activity or
the DNA-dependent RNA polymerase activity alone was inhibited, qX174 parental RF DNA synthesis occurred at near-normal efficiency. However, when the dnaC protein activity and the DNA-dependent RNA polymerase activity were simultantously inhibited, the synthesis of 4X174 parental RF DNA was 90% inhibited. In addition, we have observed that the parental RF DNA synthesized at 30 C in LD332 in the presence and absence of rifampin is stable to further incubation at 41 C (data not shown). This observation negates the possibility that parental RF DNA might have been synthesized
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FRACTION NUMBER FIG. 1. Zone sedimentation of intracellular phage DNA extracted from 0X1 74 am3-infected LD332 (dnaCt8, rifampin sensitive). The experimental details are outlined in the text. (A) Incubated and infected at 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin. The three major 32P-labeled bands in each gradient correspond to 27S single-stranded phage DNA, 21S supercoiled double-stranded RFI DNA, and 16S relaxed double-stranded RFII DNA. Sedimentation was from right to left. Unincorporated ['H]thymidine was found at the top of each gradient.
at 41 C in rifampin under the conditions described in Fig. 1 and then degraded or chased into a faster-sedimenting species. The small amount of 3H label found at the position of 3"P-labeled single-stranded DNA in Fig. 1B, C, and D has not been further examined. In other experiments where there was considerably less "2P label at this position, no 'H label was detected. In the experiment presented here, it was present in about the same amounts when parental RF synthesis was normal and when it was 90% inhibited, as well as in the absence of rifampin and in its presence, where protein synthesis was inhibited. Since RF DNA serves as the template for single-stranded virus DNA synthesis and since newly synthesized proteins are also essential for singlestranded DNA synthesis, this 'H label probably does not represent newly synthesized singlestranded DNA. We cannot exclude the possibil-
ity that it represents short segments of complementary-strand DNA on the infecting viralstrand templates.
DISCUSSION These data show that the host dnaC protein is required for OX174 parental RF DNA synthesis, presumably in the initiation of the synthesis of the complementary DNA strand, when rifampin is added at a concentration that inhibits the DNA-dependent RNA polymerase activity of the host. It is not required when rifampin is absent. In the rifampin-resistant host, whose DNA-dependent RNA polymerase is presumably resistant to rifampin, the dnaC protein is not required when rifampin is added. We conclude therefore that the DNA-dependent RNA polymerase activity of the host cell obviates the need for the dnaC protein. DNA-dependent
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J. VIROL.
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FRACTION NUMBER FIG. 2. Zone sedimentation of intracellular phage DNA from OX1 74 am3-infected LD3321 (dnaC+, rifampin sensitive). (A) 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin.
RNA polymerase catalyzes the synthesis of RNA chains on DNA templates. This RNA synthesis might obviate the need for the dnaC protein by directly providing a primer for DNA synthesis. Alternatively, the RNA may play a structural role in the initiation of DNA synthesis, thereby obviating the need for the dnaC protein. Finally, the DNA-dependent RNA polymerase may function not by synthesizing an RNA molecule, but as a structural component in an initiation complex. It seems clear that the RNA polymerase is not needed for mRNA synthesis since no new protein synthesis is required for OX174 parental RF synthesis (11). We cannot conclude that RNA polymerase and the dnaC protein catalyze the same reaction in the initiation of DNA synthesis, and that one protein can substitute directly for the other. RNA polymerase certainly cannot substitute for the dnaC protein in the semiconservative replication of the double-stranded replicative form of OX174 DNA (Fig. 1B; ref. 5), the second stage in kX174 DNA synthesis. The specificities of the two proteins must therefore be different. The same final products appear to be made no matter which of the two proteins is used in
the initiation of OX174 parental RF DNA synthesis. The OX174 RFI DNA molecules made at 30 C in the presence of rifampin and those made at 41 C in the absence of rifampin are supercoiled and stable to alkali, indicating that the DNA chains are covalently closed and free of ribose-containing nucleotides that might have been used in initiation by RNA polymerase as well as by the dnaC protein (data not shown). However, initiation might occur at a different site by a different mechanism in each case. Although DNA-dependent RNA polymerase can obviate the requirement for the dnaC protein in OX174 parental RF synthesis in vivo, the host dnaB and dnaG proteins are absolutely required (3, 6). The in vivo role of DNA-dependent RNA polymerase in this DNA synthesis is thus not simply a catalysis of random RNA primers that are elongated by DNA polymerase III assisted by the DNA unwinding protein, a pathway that is effective in vitro (4, 13, 18). Instead, the data suggest that DNA-dependent RNA polymerase substitutes only for the dnaC protein in the initiation of DNA synthesis, and that the dnaB and dnaG proteins are essential for the synthesis of the product DNA strand.
INHIBIrrION OF OX174 DNA SYNTHESIS
VOL. 16, 1975
579
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FRACTION NUMBER FIG. 3. Zone sedimentation of intracellular phage DNA from 4X174 am3-infected LD332 (dnaCt8, rifampin resistant). (A) 30 C; (B) 41 C; (C) 30 C in rifampin; (D) 41 C in rifampin.
Indeed, the two different initiation proteins probably participate in the same 4X174-specific parental RF DNA synthesis pathway now reconstituted in vitro in which the host dnaB, dnaG, and dnaE proteins play essential roles (7, 17). The participation of DNA-dependent RNA polymerase in the initiation of this OX174-specific parental RF DNA synthesis pathway in vitro has not yet been demonstrated. In fact, this enzyme can be specifically inhibited with respect to its ability to synthesize random RNA primers in the initiation of XX174 DNA synthesis in vitro. This inhibition requires the combined action of the DNA unwinding protein and a small protein that associates with the RNA polymerase (4, 19). However, our data suggest that in the cell there is sufficient DNA-dependent RNA polymerase activity to catalyze a more specific initiation of kX174 parental RF DNA synthesis. ACKNOWLEDGMENTS 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. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 2. Denhardt, D. T., and R. L. Sinsheimer. 1965. The process of infection with bacteriophage 4X174. III. Phage maturation and lysis after synchronized infection. J. Mol. Biol. 12:641-646. 3. Dumas, L. B., and C. A. Miller. 1974. Inhibition of bacteriophage 4X174 DNA replication in dnaB mutants of Escherichia coli C. J. Virol. 14:1369-1379. 4. Hurwitz, J., S. Wickner, and M. Wright. 1973. Studies on in vitro DNA synthesis. II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E. coli. Biochem. Biophys. Res. Commun. 51:257-267. 5. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophage 4X174 DNA in a temperature-sensitive dnaC mutant of Escherichia coli C. J. Virol. 13: 146-154. 6. McFadden, G., and D. T. Denhardt. 1974. Mechanism of replication of OX174 single-stranded DNA. IX. Requirement for the Escherichia coli drnG protein. J. Virol. 14:1070-1075. 7. Schekman, R., A. Weiner, and A. Kornberg. 1974. Multienzyme systems of DNA replication. Science 186:987-993.
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8. Schekman, R., W. Wickner, 0. Westergaard, D. Brutlag, K. Geider, L. L. Bertsch, and A. Kornberg. 1972. Initiation of DNA synthesis: synthesis of OX174 replicative form requires RNA synthesis resistant to rifampicin. Proc. Natl. Acad. Sci. U.S.A. 69:26912695. 9. Schubach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escherichia coli. J. Mol. Biol. 74:205-221. 10. Silverstein, S., and D. Billen. 1971. Transcription: role in the initiation and replication of DNA synthesis in Escherichia coli and OX174. Biochim. Biophys. Acta 247:383-390. 11. Sinsheimer, R. L., R. Knippers, and T. Komano. 1968. Stages in the replication of bacteriophage OX174 in vivo. Cold Spring Harbor Symp. Quant. Biol. 33: 443-447. 12. Sippel, A., and G. Hartmann. 1968. Mode of action of rifampicin on the RNA polymerase reaction. Biochim. Acta 157:218-219. 13. Tobak, H. F., J. Griffith, K. Geider, H. Schaller, and A.
14.
15.
16.
17.
18.
Kornberg. 1974. Initiation of deoxyribonucleic acid synthesis. VII. A unique location of the gap in the M13 replicative duplex synthesized in vitro. J. Biol. Chem. 249:3049-3054. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutants temperature sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. Wehrli, W., J. Nuesch, F. Knusel, and M. Staehelin. 1968. Action of rifampicins on RNA polymerase. Biochim. Biophys. Acta 157:215-217. Wickner, R. B., M. Wright, S. Wickner, and J. Hurwitz. 1972. Conversion of OX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:3233-3237. Wickner, S., and J. Hurwitz. 1974. Conversion of OX174 viral DNA to double-stranded form by purified Escherichia coli proteins. Proc. Natl. Acad. Sci. U.S.A. 71:4120-4124. Wickner, W., and A. Kornberg. 1974. A novel form of RNA polymerase from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:4425-4428.
