Vol. 18, No. 2
JouRNAL oF VImoLoGY, May 1976, p. 426-435
Printed in U.SA.
Copyright X 1976 American Society for Microbiology
Bacteriophage 4X174 Single-Stranded Viral DNA Synthesis in Temperature-Sensitive dnaB and dnaC Mutants of Escherichia coli LAWRENCE B. DUMAS* AND CHRISTINE A. MILLER Department of Biochemistry and Molecular Biology, Northwestern University Evanston, Illinois 60201 Received for publication 8 December 1975
We asked if 4X174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chloramphenicol that had been added at various times after infection to specifically stop this stage of 4X174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of 4X174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of 4X174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of 4X174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, OX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of 4X174 DNA synthesis are not necessarily mutually exclusive.
The Escherichia coli dnaC protein is required for the initiation of cycles ofchromosome replication in vivo (1, 10), whereas the dnaB protein is essential for continued DNA synthesis once the primary initiation event has occurred (4, 12). The dnaB and dnaC proteins participate in the initiation of the synthesis of the complementary strand of bacteriophage OX174 on the single-stranded (SS) viral DNA template in vitro (9, 13). Both the dnaB and dnaC proteins are essential for 4X174 DNA synthesis in vivo (3, 6). The synthesis of this phage DNA occurs in three stages (11). Neither protein is directly involved in the continuation of OX174 SS DNA synthesis, the last of the three stages, once it has begun (3, 6). However, both of these host proteins are essential for the replication of 4X174 replicative form (RF) DNA (3, 6), the template for late SS DNA synthesis. These two proteins are therefore indirectly essential for OX174 SS DNA synthesis. We asked if either the dnaB or the dnaC protein of E. coli is directly essential for the initiation of 4X174 SS DNA synthesis. We present evidence here indicating that neither protein is required. MATERIALS AND METHODS Bacteria and phage strains. LD301 (uvrA-,
thyA-, endI-, dnaEis), LD311 (uvrA-, thyA-, endI-,
dnaB's), and LD332 (uvrA-, thyA-, endL-, dnaCls) are temperature-sensitive mutants of H502 (uvrA-, thyA-, endI-) isolated in our laboratory (2, 3, 6). Stocks of 4X174 am3 (gene E, lysis defective) were prepared using E. coli C as host. Rate of X174 DNA synthesis measurements. A 200-ml culture of bacteria was grown at 30 C to a cell density of 4 x 108 cells per ml in TPGA medium (2) supplemented with 2 ,ug of thymine per ml. The cells were collected and treated with mitomycin C as previously described (3). After suspension in 10 ml of TPGA medium plus thymine, 4X174 am3 was added at a multiplicity of infection of 3. After 5 min at 30 C the culture was diluted to 200 ml with the same medium (zero time). At various times after infection portions of this culture were shifted to 41 C. In some experiments chloramphenicol was added at a final concentration of 30 Ag per ml at the time ofthe shift. The drug was removed 45 min later by filtration at 41 C. At regular intervals 2-ml portions of the cultures were pulse labeled with 5 ,uCi of [3Hlthymidine for 2 min. The pulses were terminated by the addition of an equal volume of prechilled acetone. The cells were collected by centrifugation and suspended in 1 ml of 50 mM sodium tetraborate-10 mM EDTA-200 .g of lysozyme per ml (pH 9). After 20 min at room temperature KOH was added to 0.3 N. The mixture was incubated overnight at 30 C. Calf thymus DNA (200 jig) and 10% trichloroacetic acid (2 ml) were added. The precipitates were collected and washed on glass-fiber filters. Radioactivity was measured in a liquid scintillation spectrometer. Sucrose density gradient analysis of qbX174 DNA. The bacteria were grown, mitomycin C treated, and
426
SYNTHESIS OF SINGLE-STRANDED OX174 DNA
VOL. 18, 1976
infected as described above. Portions (20 ml) of the culture were pulse-labeled for 2 min with 100 ,uCi of [3H]thymidine. The cells were collected by centrifugation, washed twice in 50 mM Tris-3 mM EDTA, pH 8, and suspended in 1 ml of the same buffer. Lysozyme was added to 200 uig per ml, and the mixture was incubated 20 min at 0 C. Sarkosyl was added to 3%, and the mixture was incubated overnight at 0 C. Protease (2 mg/ml, Sigma type VI) was added, and the mixture was incubated for 8 h at 37 C. The samples were then heated for 20 min at 56 C. 32P-labeled 4X174 viral DNA marker was added. The samples were then layered onto 36-ml linear gradients of 5 to 20% sucrose in 50 mM potas-o sium phosphate-2 mM EDTA-1 M NaCl, pH 7. These were spun 16 h at 10 C at 24,000 rpm in a Beckman SW27 rotor. Fractions were collected from the bottoms of the tubes into 1-dram glass shell vials. Radioactivity was measured in a liquid scintillation
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Rate of 4X174 DNA synthesis in the parent and mutant hosts. We measured the rates of
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80 120 160 TIME (min) FIG. 2. Rates of XX1 74 DNA synthesis at 30 and 41 C in host strain LD332 (dnaCls). At 0, 30, 60, and 75 min after infection, portions of the culture were shifted from 30 to 41 C. Symbols: *, rate at30 C; 0, rate at 41 C, shifted at 0 min; A, rate at 41 C, shifted at 30 min; , rate at 41 C, shifted at 60 min; V, rate at 41 C, shifted at 75 min.
1 | it \ am3 lysiss defective) DNA synthesis in faX174 ° host cells at 30 C and after shifting to 41 C. The
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infection by measuring the amounts of 0 1lX t [3H]thymidine incorporated into small portions ry \ of the C-treated infected cell culture Ad Ad mitomycin * 842 E I during 2-min pulses. The results from a OX174b. infected culture of the nondefective parental w 0 I host strain H502 are shown in Fig. 1. At 30 C ,w # I the rate of OX174 increased continuously for "'"' % and about 40 min after infection and then slowly t2 r > & Ad /p. % d declined. Most of the DNA synthesized at 30 C after approximately 20 to 30 min postinfection t 1/ X V in this kind of experiment was SS DNA (see ,41 below). Portions of the culture were shifted to _j |,41 C at 0, 15, 30, and 45 min after infection. The 0-40 80 120 maximum rate of OX174 SS DNA synthesis observed at 41 C was at least as high as that at TIME (min) 30 C, no matter when the culture was shifted to FIG. 1. Rates of (*Xl174 DNA synthesis at 30 and the higher temperature. the r e rac p b e n 41 C in the parent host strain H502. At 0, 15, 30 and The results from a comparable experiment 45 min after infection portions of the culture wereenwe shifted from 30 to 41 C. Symbols: 0, rate at 30 C; 0, using a X174-infected temperature-sensitive rate at 41 C, shifted at 0 min; A, rate at 41 C, shifted dnaC mutant host culture are shown in Fig. 2. at 15 min; O, rate at 41 C, shifted at 30 min; V, rate SS DNA synthesis began at approximately 30 min postinfection in this mutant at 30 C, and at 41 C, shifted at 45 min. %
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428
J. VIROL.
DUMAS AND MILLER
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TIME (min) FIG. 3. Rates of 4X1 74 DNA synthesis at 30 and 41 C in the parent host strain H502. At 10, 30, and 50 min after infection, chloramphenicol (30 HgIml) was added to 50-ml portions of the culture. These same portions were then shifted to 41 C. At 55, 75, and 95 min, respectively, the drug was removed by filtration at 41 C. Symbols: V, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 10 min, chloramphenicol removed at 55 min; 0, rate at 30 C after removal of chloramphenicol at 55 min; A, rate at 41 C, shifted at 30 minm chloramphenicol removed at 75 min; A, rate at 30 C after removal of chloramphenicol at 75 min; O, rate at 41 C, shifted at 50 min, chloramphenicol removed at 95 min; *, rate at 30 C after removal of chloramphenicol at 95 min. most of the DNA synthesized at 30 C after ap- ation of SS synthesis. The experiments reproximately 40 to 60 min postinfection was SS quired the addition of 30 ,.tg of chloramphenicol DNA (see below). At 30 C the rate of SS DNA per ml to the infected cell cultures at various synthesis increased continuously for about 2.5 h times after infection with simultaneous shifts after infection. The slower, more prolonged to 41 C. After this treatment, SS DNA synthesynthesis was probably due to the much slower sis already begun proceeds at a continuously rate of RF template DNA replication in the decreasing rate as the viral proteins, the synmutant host (6; see below). Shifts to 41 C, the thesis of which is inhibited by the chloramnonpermissive temperature for the dnaC pro- phenicol, are used up (7). Eventually SS syntein activity, inhibited SS DNA synthesis, es- thesis ceases (7). In normal host cells, RF replipecially when carried out early in infection. cation continues after the addition of chloramSimilar observations were made using the phenicol (5, 7). In the dnaB and dnaC mutant hosts RF replication is inhibited due to the dnaB mutant host (3; see below). Reinitiation of single-stranded DNA syn- temperature shift (3, 6). In our experiments the thesis. The sensitivity of 4X174 DNA synthesis chloramphenicol was later removed by filtrato early temperature shifts in the dnaB and tion at 41 C. We then monitored the rate of dnaC mutants could be simply due to inhibition pX174 DNA synthesis to see if SS synthesis of the synthesis of RF DNA molecules (3, 6), could reinitiate at 41 C. which act as templates for SS DNA synthesis, Figure 3 shows the results of the control exor to the direct inhibition of the initiation of the periment using the parent host strain. The inSS DNA synthesis stage, or both. Neither the fected cells were shifted to 41 C at 10, 30, and 50 dnaB nor the dnaC mutation directly affects SS min after infection, with simultaneous addition of chloramphenicol to 30 tmg/ml. After 45 min at synthesis once it has begun (3, 6). We tested the possibility that the dnaB and 41 C the cultures were filtered. The cells were dnaC mutations might directly affect the initi- suspended in medium without chlorampheni-
VOL. 18, 1976
0~
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SYNTHESIS OF SINGLE-STRANDED OX174 DNA
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FIG. 4. Zone sedimentation ofpulse-labeled intracellular 4X1 74 DNA extracted from parent host strain H502. At 25 min after infection a portion ofthe culture was pulse-labeled at 30 C (A). At 30 min after infection the remainder of the culture was shifted to 41 C, and chloramphenicol was added at 30 pg/ml. At 70 min a portion of the culture was pulse-labeled at 41 C (B). At 75 min after infection the remainder ofthe culture was filtered at 41 C. Half was further incubated at 41 C, and half was further incubated at 30 C. At 110 min a portion of the 41 C culture was pulse-labeled (C). At 140 min a portion of the 30 C culture was pulse-labeled (D). The arrows represent the positions of the added 32P-labeled 0X1 74 virus DNA marker.
430
J. VIROL.
DUMAS AND MILLER
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TIME (min) FIG. 5. Rates of XX1 74 DNA synthesis at 30 and 41 C in host strain LD311 (dnaB's). At 10, 30, and 50 min after infection, chloramphenicol was added to portions of the culture at a final concentration of 30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts, the chloramphenicol was removed by filtration at 41 C. Symbols: *, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 10 min, chloramphenicol removed at 55 min; A, rate at 41 C, shifted at 30 min, chloramphenicol removed at 75 min; O, rate at 41 C, shifted at 50 min, chloramphenicol removed at 95 min.
col. In all cases SS synthesis reinitiated at 41 C. SS synthesis also reinitiated at 30 C, but the same maximum rates were not achieved during the time observed. When chloramphenicol was added back to the medium after filtration, SS synthesis failed to reinitiate (data not shown). Figure 4 shows the zone sedimentation analyses of the OX174 DNA synthesized in another culture at various times during this kind of experiment. Three distinct viral DNA species could be seen in these analyses: SS DNA and the two double-stranded forms RFI and RFII (closed, circular supercoils and open, relaxed circles, respectively). These three species sediment at rates of 27, 21, and 16S, respectively. The radioactivity at the top of each gradient represents unincorporated [3H]thymidine. Immediately before the temperature shift and the addition of chloramphenicol, pulse-label was found in SS DNA and in RF DNA (Fig. 4A). Just before the removal ofthe chloramphenicol, pulse-label was found in RF DNA (Fig. 4B). But little or no label was found in SS DNA. After the removal of the drug pulse-label incorporated at 41 and 30 C was found predominantly in SS DNA (Fig. 4C and D, respectively). These data confirm the results expected: addition of chloramphenicol caused the
eventual cessation of SS DNA synthesis, whereas removal of the drug allowed SS synthesis to reinitiate. Figures 5 and 6 show the results of comparable experiments using the dnaB mutant host. Figure 5 shows that the simultaneous shift to 41 C and addition of chloramphenicol resulted in near complete inhibition of DNA synthesis. Removal of the chloramphenicol allowed reinitiation of 4X174 SS DNA synthesis at 41 C. Figure 6A shows that the 25 min after infection in another.culture at 30 C, immediately prior to a temperature shift, SS DNA was being synthesized. Immediately prior to the removal of chloramphenicol at 41 C no detectable SS DNA synthesis was occurring (Fig. 6B). After removal of the chloramphenicol, SS DNA synthesis was observed at 41 and 30 C (Fig. 6C and D, respectively). These data show that reinitiation of SS DNA synthesis occurs at 41 C in the dnaB mutant at approximately the same efficiency as in the parent host strain. The dnaB protein is therefore not essential for the reinitiation of SS DNA synthesis. Figures 7 and 8 show the results of comparable experiments using the dnaC mutant host. Again removal of the chloramphenicol allowed reinitiation of SS DNA synthesis at 41 C (Fig.
SYNTHESIS OF SINGLE-STRANDED OX174 DNA
VOL. 18, 1976
431
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The
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J. VIROL.
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TIME (min) FIG. 7. Rates of qX1 74 DNA synthesis at 30 and 41 C in host strain LD332 (dnaC"s). At 60, 80, and 100 min after infection, chloramphenicol was added to portions of the culture at a final concentration of30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts the drug was removed by filtration at 41 C. Portions (2 ml) of the cultures were pulse-labeled with 10 juCi of thymidine, twice as much as in the analogous experiments described above. Symbols: 0, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 60 min, chloramphenicol removed at 105 min; A, rate at 41 C, shifted at 80 min, chloramphenicol removed at 125 min; rate at 41 C, shifted at 100 minm chloramphenicol removed at 145 min. E,
7). Immediately prior to the removal of the drug, SS DNA synthesis was not detectable in another culture (Fig. 8B), whereas after its removal SS DNA was synthesized at a relatively rapid rate at 41 C (Fig. 80). At 30 C SS DNA synthesis was very slow (Fig. 8D). Since the reinitiation of SS DNA synthesis occurs at 41 C as efficiently as in the parent host strain, we conclude that the dnaC protein is not essential for the reinitiation of SS DNA synthesis. Previous investigations showed that OXi74 SS DNA synthesis was inhibited at 41 C late in infection in a temperature-sensitive dnaE mutant host (2) and in a dnaG mutant host (8). When the ability to resume SS DNA synthesis at 41 C was examined in the dnaE mutant in experiments comparable to those described above, we found that it was inhibited at 41 C
(Fig. 9). SS DNA synthesis resumed at 30 C in this mutant and at both temperatures in a temperature-insensitive revertant (data not shown). The same observations were made using the dnaG mutant host (data not shown). These experiments serve as controls, showing that under these conditions mutant hosts defective in late SS DNA synthesis are unable to resume SS DNA synthesis at 41 C. RF template DNA accumulation during X174 infection. Since the dnaB and dnaC proteins are not required for the reinitiation of nor for the continuation of SS DNA synthesis once it has begun, the most likely explanation for the observed sensitivity of SS synthesis to early shifts to 41 C in these mutants is the indirect effect of the inhibition of RF template DNA synthesis. Additional observations concerning 4X174 RF and SS DNA synthesis in the dnaC mutant host are consistent with this conclusion. We uniformly labeled 4X174 DNA in infected cells throughout infection with [3H]thymine and followed the accumulation of label into RF and SS DNA (distinguished by zone sedimentation). Previous experiments had shown that the rate of 45X174 RF DNA replication in a dnaC mutant host at 30 C was 15-fold less than the rate in a temperature-insensitive revertant (6). Similarly, the data in Fig. 10 show that the accumulation of [3H]thymine label into RF DNA in the dnaC mutant LD332 at 30 C was about 20-fold slower than in the temperature-insensitive revertant and the parent host strain (compare Fig. 10C to 10B and 10A, respectively; note the scale change in Fig. 10C). The rate of SS DNA synthesis was also proportionately slower. In addition, Fig. 10C shows that RF DNA synthesis continued for at least 90 min after infection at 30 C in the dnaC mutant host. Other experiments showed that RF DNA continued to be synthesized at the initial rate for at least 2 h after infection. In contrast, RF DNA synthesis occurred rapidly early in infection in the nondefective hosts, and slowed down late in infection. These data are consistent with the conclusion that the RF template concentration is limiting throughout the infection in the dnaC mutant host. Shifts to 41 C limit further RF DNA synthesis. Thus, the earlier the shift to 41 C, the less the concentration of RF template DNA, the slower the rate of SS DNA synthesis. The same explanation seems most likely for the sensitivity of SS DNA synthesis to early shifts to 41 C in the dnaB mutant. DISCUSSION Previous experiments have shown that the host cell dnaB and dnaC proteins are not
VOL. 18, 1976
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SYNTHESIS OF SINGLE-STRANDED
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FIG. 8. Zone sedimentation of pulse-labeled intracellular 4X1 74 DNA extracted from LD332 (dnaC's). This experiment was similar to that described in the legend to Fig. 4, except that the pulse included 200 10Ci of [3H]thymidine rather than 100 MCi, and the chloramphenicol was added (and the temperature shifted up) at 80 min after infection and removed at 120 min. (A) Pulse-labeled at 30 C at 75 min; (B) pulse-labeled at 41 C at 115 min; (C) pulse-labeled at 41 C at 150 min; (D) pulse-labeled at 30 C at 190 min.
needed for the continuation of SS DNA synthe- lication in the mutant cells but not in the parsis once it has begun (3, 6). The experiments ent host cells. Upon removal of the drug, SS described here show that SS DNA synthesis can DNA is made at near normal rates. These critereinitiate at 41 C in the nondefective parent host strain H502 and in the temperature-sensitive dnaB and dnaC mutant host strains. Prior to the removal of the chloramphenicol from these infected mutant cells, which triggers the reinitiation of SS DNA synthesis, SS DNA synthesis is not detectable. The elevated temperature (41 C) inactivates the temperature-sensitive proteins, resulting in inhibition of RF rep-
ria indicate that the reinitiation of SS DNA synthesis in our experiments is analogous to the normal initiation of the SS DNA synthesis stage. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not required for the initiation of SS DNA synthesis. We observed in our experiments with the mutant hosts that the maximum rates of SS
434
J. VIROL.
DUMAS AND MILLER
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rITIME (min) FIG. 9. Rates of OX1 74 DNA synthesis at 30 and 41 C in host strain LD301 (dnaEts). At 30 and 60 min after infection, chloramphenicol was added to portions of the culture at a final concentration of 30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts the drug was removed by filtration at 41 C. Portions (2 ml) were pulse-labeled with 10 uCi of 3H-thymidine. Symbols: *, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 30 min, chloramphenicol removed at 75 min; A, rate at 41 C, shifted at 60 min, chloramphenicol removed at 105 min. I
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TIME (min) FIG. 10. Relative amounts of OX1 74 RF DNA and SS DNA synthesized at 30 C in H502, in a spontaneous temperature-insensitive revertant of LD332, and in LD332. Mitomycin C-treated infected cells were uniformly labeled with [3H]thymine (10 MACi/ml) at 30 C beginning at the time of infection. At the indicated times cells from 20-ml portions were collected, washed, lysed, protease digested, and subjected to zone sedimentation in sucrose density gradients. The amounts of radioactivity in the SS and RF DNA bands were determined and normalized to the amount in SS DNA in H502 after 120 min. A, H502; B, spontaneous temperatureinsensitive revertant of LD332; C, LD332. Symbols: *, RF DNA; A, SS DNA.
DNA synthesis after reinitiation are greater the later the simultaneous shift to 41 C and addition of chloramphenicol. RF replication continues at 30 C throughout the time period
which these temperature shifts were executed. Thus the later the shift, the greater the concentration of RF DNA template. The observed increase in maximum rates of SS DNA over
VOL. 18, 1976
SYNTHESIS OF SINGLE-STRANDED
OX174
DNA
435
synthesis is consistent with the increase in tem- sion of RF DNA replication once SS DNA synplate concentration. thesis has begun. We attempted to add the chloramphenicol at ACKNOWLEDGMENTS the time of infection in experiments similar to was supported by Public Health Service This work those described here (data not shown). Under research grant AI-9882 and research career development these conditions no appreciable SS DNA syn- award AI-70,632 (to L.B.D.) from the National Institute of thesis would have occurred prior to the removal Allergy and Infectious Diseases. of the drug. We found that cells that are kept at LITERATURE CITED 30 C can achieve normal rates of SS synthesis 1970. Escherichia coli mutants with temper1. P. L. Carl, upon removal of the drug, but that RF replicaature-sensitive synthesis of DNA. Mol. Gen. Genet. tion is required after removal of the drug to 109:107-122. achieve these rates. RF replication is slow at 2. Dumas, L. B., and C. A. Miller. 1973. Replication of bacteriophage 4X174 DNA in a temperature-sensi30 C in chloramphenicol. Upon shifting to 41 C, tive dnaE mutant of Escherichia coli C. J. Virol. at the time of removal of the chloramphenicol, 11:848-855. we observed only slow SS DNA synthesis lim- 3. Dumas, L. B., and C. A. Miller. 1974. Inhibition of bacteriophage 4X174 DNA replication in dnaB muited by the concentration of RF template DNA. tants of Escherichia coli C. J. Virol. 14:1369-1379. To allow accumulation of enough RF template 4. Hirota, Y., A. Ryter, and F. Jacob. 1968. ThermosensiDNA to achieve normal rates of SS DNA syntive mutants of E. coli affected in the processes of thesis without further RF replication, we added DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33:677-694. the chloramphenicol late enough such that SS 5. Hutchison, C. A., and R. L. Sinsheimer. 1966. The DNA synthesis had begun. process of infection with bacteriophage 4X174. X. We conclude from these observations that the Mutations in a XX lysis gene. J. Mol. Biol. 18:429dnaB and dnaC proteins are not directly essen447. tial for either the initiation of SS DNA synthe- 6. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophage 4bX174 DNA in a temperature-sensisis or its continuation. The reduced rates of SS tive dnaC mutant of Escherichia coli C. J. Virol. DNA synthesis observed at 41 C in these mu13:146-154. tant cells, especially when the temperature was 7. Levine, A. J., and R. L. Sinsheimer. 1969. The process of infection with bacteriophage X174. XXV. Studies shifted up to 41 C early in infection, are most with bacteriophage 4oX174 mutants blocked in proglikely due to inhibition of the synthesis of the eny replicative form DNA synthesis. J. Mol. Biol. RF DNA template needed for SS DNA synthe39:619-639. sis. 8. McFadden, G., and D. T. Denhardt. 1974. Mechanism of replication of 4X174 single-stranded DNA. IX. ReThe experiments described here also show quirement for the Escherichia coli dnaG protein. J. that RF DNA replication does not necessarily Virol. 14:1070-1075. cease at the time of the initiation of SS DNA 9. Schekman, R., A. Weiner, and A. Kornberg. 1974. Mulsynthesis (10). In normal host cells most of the tienzyme systems of DNA replication. Science 186:987-993. RF DNA is synthesized early in infection, and W. H., J. D. Whitmer, and C. I. Davern. most of the SS DNA is synthesized late. How- 10. Schubach, 1973. Genetic control of DNA initiation in Escherever, in the dnaC mutant cells RF DNA repliichia coli. J. Mol. Biol. 74:205-221. cation continues at its early rate for at least 2 h 11. Sinsheimer, R. L., R. Knippers, and T. Komano. 1968. Stages in the replication of bacteriophage 4X174 in after infection, whereas SS DNA synthesis bevivo. Cold Spring Harbor Symp. Quant. Biol. 33:443gins as early as 30 min after infection. RF 447. replication and SS synthesis are not therefore 12. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutually exclusive. Rather, in normal infecmutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. tions the saturating level of RF DNA is apparS., and J. Hurwitz. 1974. Conversion of qbX174 ently achieved early in infection, at about the 13. Wickner, viral DNA to double-stranded form by purified Eschsame time as the onset of SS DNA synthesis. erichia coli proteins. Proc. Natl. Acad. Sci. U.S.A. But there does not seem to be a necessary exclu71:4120-4124.
JouRNAL oF VImoLoGY, May 1976, p. 426-435
Printed in U.SA.
Copyright X 1976 American Society for Microbiology
Bacteriophage 4X174 Single-Stranded Viral DNA Synthesis in Temperature-Sensitive dnaB and dnaC Mutants of Escherichia coli LAWRENCE B. DUMAS* AND CHRISTINE A. MILLER Department of Biochemistry and Molecular Biology, Northwestern University Evanston, Illinois 60201 Received for publication 8 December 1975
We asked if 4X174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chloramphenicol that had been added at various times after infection to specifically stop this stage of 4X174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of 4X174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of 4X174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of 4X174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, OX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of 4X174 DNA synthesis are not necessarily mutually exclusive.
The Escherichia coli dnaC protein is required for the initiation of cycles ofchromosome replication in vivo (1, 10), whereas the dnaB protein is essential for continued DNA synthesis once the primary initiation event has occurred (4, 12). The dnaB and dnaC proteins participate in the initiation of the synthesis of the complementary strand of bacteriophage OX174 on the single-stranded (SS) viral DNA template in vitro (9, 13). Both the dnaB and dnaC proteins are essential for 4X174 DNA synthesis in vivo (3, 6). The synthesis of this phage DNA occurs in three stages (11). Neither protein is directly involved in the continuation of OX174 SS DNA synthesis, the last of the three stages, once it has begun (3, 6). However, both of these host proteins are essential for the replication of 4X174 replicative form (RF) DNA (3, 6), the template for late SS DNA synthesis. These two proteins are therefore indirectly essential for OX174 SS DNA synthesis. We asked if either the dnaB or the dnaC protein of E. coli is directly essential for the initiation of 4X174 SS DNA synthesis. We present evidence here indicating that neither protein is required. MATERIALS AND METHODS Bacteria and phage strains. LD301 (uvrA-,
thyA-, endI-, dnaEis), LD311 (uvrA-, thyA-, endI-,
dnaB's), and LD332 (uvrA-, thyA-, endL-, dnaCls) are temperature-sensitive mutants of H502 (uvrA-, thyA-, endI-) isolated in our laboratory (2, 3, 6). Stocks of 4X174 am3 (gene E, lysis defective) were prepared using E. coli C as host. Rate of X174 DNA synthesis measurements. A 200-ml culture of bacteria was grown at 30 C to a cell density of 4 x 108 cells per ml in TPGA medium (2) supplemented with 2 ,ug of thymine per ml. The cells were collected and treated with mitomycin C as previously described (3). After suspension in 10 ml of TPGA medium plus thymine, 4X174 am3 was added at a multiplicity of infection of 3. After 5 min at 30 C the culture was diluted to 200 ml with the same medium (zero time). At various times after infection portions of this culture were shifted to 41 C. In some experiments chloramphenicol was added at a final concentration of 30 Ag per ml at the time ofthe shift. The drug was removed 45 min later by filtration at 41 C. At regular intervals 2-ml portions of the cultures were pulse labeled with 5 ,uCi of [3Hlthymidine for 2 min. The pulses were terminated by the addition of an equal volume of prechilled acetone. The cells were collected by centrifugation and suspended in 1 ml of 50 mM sodium tetraborate-10 mM EDTA-200 .g of lysozyme per ml (pH 9). After 20 min at room temperature KOH was added to 0.3 N. The mixture was incubated overnight at 30 C. Calf thymus DNA (200 jig) and 10% trichloroacetic acid (2 ml) were added. The precipitates were collected and washed on glass-fiber filters. Radioactivity was measured in a liquid scintillation spectrometer. Sucrose density gradient analysis of qbX174 DNA. The bacteria were grown, mitomycin C treated, and
426
SYNTHESIS OF SINGLE-STRANDED OX174 DNA
VOL. 18, 1976
infected as described above. Portions (20 ml) of the culture were pulse-labeled for 2 min with 100 ,uCi of [3H]thymidine. The cells were collected by centrifugation, washed twice in 50 mM Tris-3 mM EDTA, pH 8, and suspended in 1 ml of the same buffer. Lysozyme was added to 200 uig per ml, and the mixture was incubated 20 min at 0 C. Sarkosyl was added to 3%, and the mixture was incubated overnight at 0 C. Protease (2 mg/ml, Sigma type VI) was added, and the mixture was incubated for 8 h at 37 C. The samples were then heated for 20 min at 56 C. 32P-labeled 4X174 viral DNA marker was added. The samples were then layered onto 36-ml linear gradients of 5 to 20% sucrose in 50 mM potas-o sium phosphate-2 mM EDTA-1 M NaCl, pH 7. These were spun 16 h at 10 C at 24,000 rpm in a Beckman SW27 rotor. Fractions were collected from the bottoms of the tubes into 1-dram glass shell vials. Radioactivity was measured in a liquid scintillation
V |
ri 16 _
12 -
l |
0
4_
Rate of 4X174 DNA synthesis in the parent and mutant hosts. We measured the rates of
10
T 2 N
A,3 ° ° % * 3% ~ I ' 5 3%
X
bfi 6U
1
yA
4 40
80 120 160 TIME (min) FIG. 2. Rates of XX1 74 DNA synthesis at 30 and 41 C in host strain LD332 (dnaCls). At 0, 30, 60, and 75 min after infection, portions of the culture were shifted from 30 to 41 C. Symbols: *, rate at30 C; 0, rate at 41 C, shifted at 0 min; A, rate at 41 C, shifted at 30 min; , rate at 41 C, shifted at 60 min; V, rate at 41 C, shifted at 75 min.
1 | it \ am3 lysiss defective) DNA synthesis in faX174 ° host cells at 30 C and after shifting to 41 C. The
I3
.C
:I
tVV ::d E i% L | / aO..2 0 W
RESULTS
8
|
N
spectrometer.
10__
427
9e8
%
\rates
were determined at various times after
infection by measuring the amounts of 0 1lX t [3H]thymidine incorporated into small portions ry \ of the C-treated infected cell culture Ad Ad mitomycin * 842 E I during 2-min pulses. The results from a OX174b. infected culture of the nondefective parental w 0 I host strain H502 are shown in Fig. 1. At 30 C ,w # I the rate of OX174 increased continuously for "'"' % and about 40 min after infection and then slowly t2 r > & Ad /p. % d declined. Most of the DNA synthesized at 30 C after approximately 20 to 30 min postinfection t 1/ X V in this kind of experiment was SS DNA (see ,41 below). Portions of the culture were shifted to _j |,41 C at 0, 15, 30, and 45 min after infection. The 0-40 80 120 maximum rate of OX174 SS DNA synthesis observed at 41 C was at least as high as that at TIME (min) 30 C, no matter when the culture was shifted to FIG. 1. Rates of (*Xl174 DNA synthesis at 30 and the higher temperature. the r e rac p b e n 41 C in the parent host strain H502. At 0, 15, 30 and The results from a comparable experiment 45 min after infection portions of the culture wereenwe shifted from 30 to 41 C. Symbols: 0, rate at 30 C; 0, using a X174-infected temperature-sensitive rate at 41 C, shifted at 0 min; A, rate at 41 C, shifted dnaC mutant host culture are shown in Fig. 2. at 15 min; O, rate at 41 C, shifted at 30 min; V, rate SS DNA synthesis began at approximately 30 min postinfection in this mutant at 30 C, and at 41 C, shifted at 45 min. %
04
-
m
428
J. VIROL.
DUMAS AND MILLER
E
o2 8
0
%~~~~~~~~~
01%
CLJ 4
%
40
80
120
160
TIME (min) FIG. 3. Rates of 4X1 74 DNA synthesis at 30 and 41 C in the parent host strain H502. At 10, 30, and 50 min after infection, chloramphenicol (30 HgIml) was added to 50-ml portions of the culture. These same portions were then shifted to 41 C. At 55, 75, and 95 min, respectively, the drug was removed by filtration at 41 C. Symbols: V, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 10 min, chloramphenicol removed at 55 min; 0, rate at 30 C after removal of chloramphenicol at 55 min; A, rate at 41 C, shifted at 30 minm chloramphenicol removed at 75 min; A, rate at 30 C after removal of chloramphenicol at 75 min; O, rate at 41 C, shifted at 50 min, chloramphenicol removed at 95 min; *, rate at 30 C after removal of chloramphenicol at 95 min. most of the DNA synthesized at 30 C after ap- ation of SS synthesis. The experiments reproximately 40 to 60 min postinfection was SS quired the addition of 30 ,.tg of chloramphenicol DNA (see below). At 30 C the rate of SS DNA per ml to the infected cell cultures at various synthesis increased continuously for about 2.5 h times after infection with simultaneous shifts after infection. The slower, more prolonged to 41 C. After this treatment, SS DNA synthesynthesis was probably due to the much slower sis already begun proceeds at a continuously rate of RF template DNA replication in the decreasing rate as the viral proteins, the synmutant host (6; see below). Shifts to 41 C, the thesis of which is inhibited by the chloramnonpermissive temperature for the dnaC pro- phenicol, are used up (7). Eventually SS syntein activity, inhibited SS DNA synthesis, es- thesis ceases (7). In normal host cells, RF replipecially when carried out early in infection. cation continues after the addition of chloramSimilar observations were made using the phenicol (5, 7). In the dnaB and dnaC mutant hosts RF replication is inhibited due to the dnaB mutant host (3; see below). Reinitiation of single-stranded DNA syn- temperature shift (3, 6). In our experiments the thesis. The sensitivity of 4X174 DNA synthesis chloramphenicol was later removed by filtrato early temperature shifts in the dnaB and tion at 41 C. We then monitored the rate of dnaC mutants could be simply due to inhibition pX174 DNA synthesis to see if SS synthesis of the synthesis of RF DNA molecules (3, 6), could reinitiate at 41 C. which act as templates for SS DNA synthesis, Figure 3 shows the results of the control exor to the direct inhibition of the initiation of the periment using the parent host strain. The inSS DNA synthesis stage, or both. Neither the fected cells were shifted to 41 C at 10, 30, and 50 dnaB nor the dnaC mutation directly affects SS min after infection, with simultaneous addition of chloramphenicol to 30 tmg/ml. After 45 min at synthesis once it has begun (3, 6). We tested the possibility that the dnaB and 41 C the cultures were filtered. The cells were dnaC mutations might directly affect the initi- suspended in medium without chlorampheni-
VOL. 18, 1976
0~
10xWI
SYNTHESIS OF SINGLE-STRANDED OX174 DNA
I
429
I
0
16-
12
8
-
4
20
40 60 20 40 FRACTION NUMBER
60
FIG. 4. Zone sedimentation ofpulse-labeled intracellular 4X1 74 DNA extracted from parent host strain H502. At 25 min after infection a portion ofthe culture was pulse-labeled at 30 C (A). At 30 min after infection the remainder of the culture was shifted to 41 C, and chloramphenicol was added at 30 pg/ml. At 70 min a portion of the culture was pulse-labeled at 41 C (B). At 75 min after infection the remainder ofthe culture was filtered at 41 C. Half was further incubated at 41 C, and half was further incubated at 30 C. At 110 min a portion of the 41 C culture was pulse-labeled (C). At 140 min a portion of the 30 C culture was pulse-labeled (D). The arrows represent the positions of the added 32P-labeled 0X1 74 virus DNA marker.
430
J. VIROL.
DUMAS AND MILLER
o o
0~~~~
.s 4E
, 9%2,t '~~~~~~~~~~~ /
2
R
~~ %%~~~~~~~~~~I I
C
\
% ~~~~.Ie
~~~40
80
~
%
120
160
TIME (min) FIG. 5. Rates of XX1 74 DNA synthesis at 30 and 41 C in host strain LD311 (dnaB's). At 10, 30, and 50 min after infection, chloramphenicol was added to portions of the culture at a final concentration of 30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts, the chloramphenicol was removed by filtration at 41 C. Symbols: *, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 10 min, chloramphenicol removed at 55 min; A, rate at 41 C, shifted at 30 min, chloramphenicol removed at 75 min; O, rate at 41 C, shifted at 50 min, chloramphenicol removed at 95 min.
col. In all cases SS synthesis reinitiated at 41 C. SS synthesis also reinitiated at 30 C, but the same maximum rates were not achieved during the time observed. When chloramphenicol was added back to the medium after filtration, SS synthesis failed to reinitiate (data not shown). Figure 4 shows the zone sedimentation analyses of the OX174 DNA synthesized in another culture at various times during this kind of experiment. Three distinct viral DNA species could be seen in these analyses: SS DNA and the two double-stranded forms RFI and RFII (closed, circular supercoils and open, relaxed circles, respectively). These three species sediment at rates of 27, 21, and 16S, respectively. The radioactivity at the top of each gradient represents unincorporated [3H]thymidine. Immediately before the temperature shift and the addition of chloramphenicol, pulse-label was found in SS DNA and in RF DNA (Fig. 4A). Just before the removal ofthe chloramphenicol, pulse-label was found in RF DNA (Fig. 4B). But little or no label was found in SS DNA. After the removal of the drug pulse-label incorporated at 41 and 30 C was found predominantly in SS DNA (Fig. 4C and D, respectively). These data confirm the results expected: addition of chloramphenicol caused the
eventual cessation of SS DNA synthesis, whereas removal of the drug allowed SS synthesis to reinitiate. Figures 5 and 6 show the results of comparable experiments using the dnaB mutant host. Figure 5 shows that the simultaneous shift to 41 C and addition of chloramphenicol resulted in near complete inhibition of DNA synthesis. Removal of the chloramphenicol allowed reinitiation of 4X174 SS DNA synthesis at 41 C. Figure 6A shows that the 25 min after infection in another.culture at 30 C, immediately prior to a temperature shift, SS DNA was being synthesized. Immediately prior to the removal of chloramphenicol at 41 C no detectable SS DNA synthesis was occurring (Fig. 6B). After removal of the chloramphenicol, SS DNA synthesis was observed at 41 and 30 C (Fig. 6C and D, respectively). These data show that reinitiation of SS DNA synthesis occurs at 41 C in the dnaB mutant at approximately the same efficiency as in the parent host strain. The dnaB protein is therefore not essential for the reinitiation of SS DNA synthesis. Figures 7 and 8 show the results of comparable experiments using the dnaC mutant host. Again removal of the chloramphenicol allowed reinitiation of SS DNA synthesis at 41 C (Fig.
SYNTHESIS OF SINGLE-STRANDED OX174 DNA
VOL. 18, 1976
431
16 12
4
to
_
%
2
I
I
I
C
16
-
12
-
D
h
8 4-
20
20 40 FRACTION NUMBER
40
60
60
FIG. 6. Zone sedimentation ofpulse-labeled intracellular MAXl 74 DNA extracted from LD311 (dnaB5). experiment was carried out exactly as described in the legend to Fig. 4.
The
432
J. VIROL.
DUMAS AND MILLER
24p
9%~~~~~~~~~~~~
0
0
16
18
~~~~~~~~~it I
0
1
060
120
180
TIME (min) FIG. 7. Rates of qX1 74 DNA synthesis at 30 and 41 C in host strain LD332 (dnaC"s). At 60, 80, and 100 min after infection, chloramphenicol was added to portions of the culture at a final concentration of30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts the drug was removed by filtration at 41 C. Portions (2 ml) of the cultures were pulse-labeled with 10 juCi of thymidine, twice as much as in the analogous experiments described above. Symbols: 0, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 60 min, chloramphenicol removed at 105 min; A, rate at 41 C, shifted at 80 min, chloramphenicol removed at 125 min; rate at 41 C, shifted at 100 minm chloramphenicol removed at 145 min. E,
7). Immediately prior to the removal of the drug, SS DNA synthesis was not detectable in another culture (Fig. 8B), whereas after its removal SS DNA was synthesized at a relatively rapid rate at 41 C (Fig. 80). At 30 C SS DNA synthesis was very slow (Fig. 8D). Since the reinitiation of SS DNA synthesis occurs at 41 C as efficiently as in the parent host strain, we conclude that the dnaC protein is not essential for the reinitiation of SS DNA synthesis. Previous investigations showed that OXi74 SS DNA synthesis was inhibited at 41 C late in infection in a temperature-sensitive dnaE mutant host (2) and in a dnaG mutant host (8). When the ability to resume SS DNA synthesis at 41 C was examined in the dnaE mutant in experiments comparable to those described above, we found that it was inhibited at 41 C
(Fig. 9). SS DNA synthesis resumed at 30 C in this mutant and at both temperatures in a temperature-insensitive revertant (data not shown). The same observations were made using the dnaG mutant host (data not shown). These experiments serve as controls, showing that under these conditions mutant hosts defective in late SS DNA synthesis are unable to resume SS DNA synthesis at 41 C. RF template DNA accumulation during X174 infection. Since the dnaB and dnaC proteins are not required for the reinitiation of nor for the continuation of SS DNA synthesis once it has begun, the most likely explanation for the observed sensitivity of SS synthesis to early shifts to 41 C in these mutants is the indirect effect of the inhibition of RF template DNA synthesis. Additional observations concerning 4X174 RF and SS DNA synthesis in the dnaC mutant host are consistent with this conclusion. We uniformly labeled 4X174 DNA in infected cells throughout infection with [3H]thymine and followed the accumulation of label into RF and SS DNA (distinguished by zone sedimentation). Previous experiments had shown that the rate of 45X174 RF DNA replication in a dnaC mutant host at 30 C was 15-fold less than the rate in a temperature-insensitive revertant (6). Similarly, the data in Fig. 10 show that the accumulation of [3H]thymine label into RF DNA in the dnaC mutant LD332 at 30 C was about 20-fold slower than in the temperature-insensitive revertant and the parent host strain (compare Fig. 10C to 10B and 10A, respectively; note the scale change in Fig. 10C). The rate of SS DNA synthesis was also proportionately slower. In addition, Fig. 10C shows that RF DNA synthesis continued for at least 90 min after infection at 30 C in the dnaC mutant host. Other experiments showed that RF DNA continued to be synthesized at the initial rate for at least 2 h after infection. In contrast, RF DNA synthesis occurred rapidly early in infection in the nondefective hosts, and slowed down late in infection. These data are consistent with the conclusion that the RF template concentration is limiting throughout the infection in the dnaC mutant host. Shifts to 41 C limit further RF DNA synthesis. Thus, the earlier the shift to 41 C, the less the concentration of RF template DNA, the slower the rate of SS DNA synthesis. The same explanation seems most likely for the sensitivity of SS DNA synthesis to early shifts to 41 C in the dnaB mutant. DISCUSSION Previous experiments have shown that the host cell dnaB and dnaC proteins are not
VOL. 18, 1976
'0
'°o~I
0~
SYNTHESIS OF SINGLE-STRANDED
I
I
OX174
DNA
433
III
4
I
20
j
-
40 60 20 40 FRACTION NUMBER
60
FIG. 8. Zone sedimentation of pulse-labeled intracellular 4X1 74 DNA extracted from LD332 (dnaC's). This experiment was similar to that described in the legend to Fig. 4, except that the pulse included 200 10Ci of [3H]thymidine rather than 100 MCi, and the chloramphenicol was added (and the temperature shifted up) at 80 min after infection and removed at 120 min. (A) Pulse-labeled at 30 C at 75 min; (B) pulse-labeled at 41 C at 115 min; (C) pulse-labeled at 41 C at 150 min; (D) pulse-labeled at 30 C at 190 min.
needed for the continuation of SS DNA synthe- lication in the mutant cells but not in the parsis once it has begun (3, 6). The experiments ent host cells. Upon removal of the drug, SS described here show that SS DNA synthesis can DNA is made at near normal rates. These critereinitiate at 41 C in the nondefective parent host strain H502 and in the temperature-sensitive dnaB and dnaC mutant host strains. Prior to the removal of the chloramphenicol from these infected mutant cells, which triggers the reinitiation of SS DNA synthesis, SS DNA synthesis is not detectable. The elevated temperature (41 C) inactivates the temperature-sensitive proteins, resulting in inhibition of RF rep-
ria indicate that the reinitiation of SS DNA synthesis in our experiments is analogous to the normal initiation of the SS DNA synthesis stage. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not required for the initiation of SS DNA synthesis. We observed in our experiments with the mutant hosts that the maximum rates of SS
434
J. VIROL.
DUMAS AND MILLER
IE C'J 6 la
0
U. 0
0
E .2 w
rITIME (min) FIG. 9. Rates of OX1 74 DNA synthesis at 30 and 41 C in host strain LD301 (dnaEts). At 30 and 60 min after infection, chloramphenicol was added to portions of the culture at a final concentration of 30 pg/ml. These same portions were simultaneously shifted to 41 C. At 45 min after the shifts the drug was removed by filtration at 41 C. Portions (2 ml) were pulse-labeled with 10 uCi of 3H-thymidine. Symbols: *, rate at 30 C, no chloramphenicol added; 0, rate at 41 C, shifted at 30 min, chloramphenicol removed at 75 min; A, rate at 41 C, shifted at 60 min, chloramphenicol removed at 105 min. I
I
B
A
0.05k
1.01-
F-0.8 z
I
II III I I I I
I I
40
'
_
II II I II I
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-_
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0.2 _
I
10.04
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1 0.4
0.0
I
14I
> 0.6
C
80
120
I
40
80
0.71 I 120
40
80
120
TIME (min) FIG. 10. Relative amounts of OX1 74 RF DNA and SS DNA synthesized at 30 C in H502, in a spontaneous temperature-insensitive revertant of LD332, and in LD332. Mitomycin C-treated infected cells were uniformly labeled with [3H]thymine (10 MACi/ml) at 30 C beginning at the time of infection. At the indicated times cells from 20-ml portions were collected, washed, lysed, protease digested, and subjected to zone sedimentation in sucrose density gradients. The amounts of radioactivity in the SS and RF DNA bands were determined and normalized to the amount in SS DNA in H502 after 120 min. A, H502; B, spontaneous temperatureinsensitive revertant of LD332; C, LD332. Symbols: *, RF DNA; A, SS DNA.
DNA synthesis after reinitiation are greater the later the simultaneous shift to 41 C and addition of chloramphenicol. RF replication continues at 30 C throughout the time period
which these temperature shifts were executed. Thus the later the shift, the greater the concentration of RF DNA template. The observed increase in maximum rates of SS DNA over
VOL. 18, 1976
SYNTHESIS OF SINGLE-STRANDED
OX174
DNA
435
synthesis is consistent with the increase in tem- sion of RF DNA replication once SS DNA synplate concentration. thesis has begun. We attempted to add the chloramphenicol at ACKNOWLEDGMENTS the time of infection in experiments similar to was supported by Public Health Service This work those described here (data not shown). Under research grant AI-9882 and research career development these conditions no appreciable SS DNA syn- award AI-70,632 (to L.B.D.) from the National Institute of thesis would have occurred prior to the removal Allergy and Infectious Diseases. of the drug. We found that cells that are kept at LITERATURE CITED 30 C can achieve normal rates of SS synthesis 1970. Escherichia coli mutants with temper1. P. L. Carl, upon removal of the drug, but that RF replicaature-sensitive synthesis of DNA. Mol. Gen. Genet. tion is required after removal of the drug to 109:107-122. achieve these rates. RF replication is slow at 2. Dumas, L. B., and C. A. Miller. 1973. Replication of bacteriophage 4X174 DNA in a temperature-sensi30 C in chloramphenicol. Upon shifting to 41 C, tive dnaE mutant of Escherichia coli C. J. Virol. at the time of removal of the chloramphenicol, 11:848-855. we observed only slow SS DNA synthesis lim- 3. Dumas, L. B., and C. A. Miller. 1974. Inhibition of bacteriophage 4X174 DNA replication in dnaB muited by the concentration of RF template DNA. tants of Escherichia coli C. J. Virol. 14:1369-1379. To allow accumulation of enough RF template 4. Hirota, Y., A. Ryter, and F. Jacob. 1968. ThermosensiDNA to achieve normal rates of SS DNA syntive mutants of E. coli affected in the processes of thesis without further RF replication, we added DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33:677-694. the chloramphenicol late enough such that SS 5. Hutchison, C. A., and R. L. Sinsheimer. 1966. The DNA synthesis had begun. process of infection with bacteriophage 4X174. X. We conclude from these observations that the Mutations in a XX lysis gene. J. Mol. Biol. 18:429dnaB and dnaC proteins are not directly essen447. tial for either the initiation of SS DNA synthe- 6. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophage 4bX174 DNA in a temperature-sensisis or its continuation. The reduced rates of SS tive dnaC mutant of Escherichia coli C. J. Virol. DNA synthesis observed at 41 C in these mu13:146-154. tant cells, especially when the temperature was 7. Levine, A. J., and R. L. Sinsheimer. 1969. The process of infection with bacteriophage X174. XXV. Studies shifted up to 41 C early in infection, are most with bacteriophage 4oX174 mutants blocked in proglikely due to inhibition of the synthesis of the eny replicative form DNA synthesis. J. Mol. Biol. RF DNA template needed for SS DNA synthe39:619-639. sis. 8. McFadden, G., and D. T. Denhardt. 1974. Mechanism of replication of 4X174 single-stranded DNA. IX. ReThe experiments described here also show quirement for the Escherichia coli dnaG protein. J. that RF DNA replication does not necessarily Virol. 14:1070-1075. cease at the time of the initiation of SS DNA 9. Schekman, R., A. Weiner, and A. Kornberg. 1974. Mulsynthesis (10). In normal host cells most of the tienzyme systems of DNA replication. Science 186:987-993. RF DNA is synthesized early in infection, and W. H., J. D. Whitmer, and C. I. Davern. most of the SS DNA is synthesized late. How- 10. Schubach, 1973. Genetic control of DNA initiation in Escherever, in the dnaC mutant cells RF DNA repliichia coli. J. Mol. Biol. 74:205-221. cation continues at its early rate for at least 2 h 11. Sinsheimer, R. L., R. Knippers, and T. Komano. 1968. Stages in the replication of bacteriophage 4X174 in after infection, whereas SS DNA synthesis bevivo. Cold Spring Harbor Symp. Quant. Biol. 33:443gins as early as 30 min after infection. RF 447. replication and SS synthesis are not therefore 12. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutually exclusive. Rather, in normal infecmutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. tions the saturating level of RF DNA is apparS., and J. Hurwitz. 1974. Conversion of qbX174 ently achieved early in infection, at about the 13. Wickner, viral DNA to double-stranded form by purified Eschsame time as the onset of SS DNA synthesis. erichia coli proteins. Proc. Natl. Acad. Sci. U.S.A. But there does not seem to be a necessary exclu71:4120-4124.
