JOURNAL OF BACrERIOLOGY, Mar. 1975, p. 883-891 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 3
Printed in U.S.A.
Accumulation of the Capacity for Initiation of Deoxyribonucleic Acid Replication in Escherichia coli IRENE M. EVANS
AND
HELEN EBERLE*
Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642 Received for publication 15 October 1974
Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents. The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited. Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis. This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved. These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation.
In the bacterial cell cycle some proteins are synthesized continuously throughout the cycle while others are synthesized only periodically (14, 18, 24). Whether a protein is available for use continuously or only at particular times may have important consequences for control if such a protein is important in regulating cell cycle events, e.g., initiation of deoxyribonucleic acid (DNA) synthesis. Attempts have been made to determine whether the products necessary for DNA initiation are made continuously during a period when cells were held at a temperature nonpermissive for DNA initiation and synthesis. By measuring residual DNA synthesis in the presence of chloramphenicol (CAP) after temperature-sensitive initiation mutants had been held at nonpermissive temperature, Abe and Tomizawa (1) and Beyersmann et al. (2) found that the [3H]thymine counts in the culture approximately doubled, suggesting that only one cycle of DNA was able to be synthesized. Further protein synthesis at the permissive temperature seemed to be necessary to permit a second round of DNA synthesis. This confirmed the earlier findings of Schwartz and Worcel (22),who used a dnaB mutant to study this question. Schubach et al. (21), however, concluded from similar experiments that products needed for DNA synthesis were able to accumulate continuously at a nonpermissive temperature, allowing more than one round of DNA
synthesis without new protein synthesis upon return to permissive temperature. Recently, Lark (12) and Messer (17) obtained evidence which shows that CAP affects DNA chain elongation. Since most of the previous estimates of the number of rounds of DNA synthesized in the presence of CAP have been calculated from the amount of residual DNA synthesis in the presence of CAP (a calculation which assumed that CAP only blocked DNA initiation), it seemed possible that the number of initiation events might have been underestimated in these earlier studies. Therefore, the number of initiation events occurring in the presence of CAP and other initiation inhibitors has been reinvestigated by measuring the rate of DNA synthesis after the release df temperature-sensitive initiation mutants from nonpermissive conditions. The results indicate that more than one round of DNA synthesis may be initiated in the presence of these inhibitors. The failure to detect such reinitiation events in previous residual synthesis experiments may be explained by the possible masking of reinitiation by premature chain termination. MATERIALS AND METHODS Bacteria. The following strains were utilized: Escherichia coli 15T-ts-489, dnaC (Thy-, Arg-, Met-, Trp-; obtained from K. G. Lark); E. coli K-12, dna252A (2) (Thi-, Thy-, Pro-, StrS; obtained from F. Bonhoeffer and called Hfr 165/120/6); E. coli K-12,
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dna2C (Thy-, Leu-, StrR; obtained from P. Carl and called PC-2) (4); E. coli K-12, dnaA (Thy-, Thi-; obtained from Tomizawa and called N-167) (1). Growth conditions. All cells were grown in M9 minimal medium (15) to which required supplements were added as needed (micrograms per milliliter): thymine, 4; proline, 20; arginine, 20; methionine, 20; tryptophan, 20; leucine, 40; and thiamine hydrochloride (vitamin B1), 2. The K-12 strains were normally grown with the addition of vitamin-free Casamino Acids (1%) (Difco). Medium changes were accomplished by collecting cells on 0.45-Mm membrane filters (Millipore Corp. or Schleicher and Schuell), washing with prewarmed M9 medium, and suspending cells in the desired medium. The doubling time for the strains Hfr 165/120/6, PC-2, and N-167 in minimal M9 media was 55 to 60 min at 30 to 32 C. The generation time for strain 15T-ts-489, grown at 30 to 32 C with the required supplements, was 45 min. In all experiments, 30 to 32 C was the permissive temperature for DNA initiation which ceased when the strains were transferred to 42 C (43 C for strain N-167). Inhibitors. All inhibitors blocked DNA synthesis within 40 to 80 min after addition. CAP (Parke, Davis and Co.), at 150 Mg/ml, inhibited protein synthesis essentially immediately, whereas 30,Mg of CAP per ml reduced protein synthesis to a level 10 to 30% that of the control. Phenethyl alcohol (PEA; Eastman Kodak Co.), at 0.3%, allowed a low level of ribonucleic acid synthesis and permitted protein synthesis at a level 20 to 40% that of the control. Rifampin (Calbiochem), at 100 Mg/ml, blocked ribonucleic acid and protein synthesis essentially immediately upon addition. CAP stock solutions were prepared fresh daily in a 47.5% ethanol solution at a concentration of 3 to 6 mg/ml. A rifampin stock solution was made by dissolving 10 mg of rifampin in methanol, and stored in the dark at 0 C. PEA was stored at room temperature and added to cultures at the necessary concentrations. Control experiments demonstrated that neither methanol nor ethanol had any effect on the amount of radioactivity incorporated into DNA under the experimental conditions at 30 to 32 C. Radioactive labeling: (i) Continuous label experiments. For experiments in which DNA synthesis was measured, [methyl-3H ]thymine (New England Nuclear) at 2 MCi/4 Mg per ml was added. Duplicate 0.1-ml samples of culture were removed and precipitated by 1 ml of 5%' cold trichloroacetic acid. Precipitates were collected on Reeve Angel 934AH glass-fiber filters (Clifton, N.J.), washed with cold distilled water, and dried, and the radioactivity was determined on a Beckman model LS250 scintillation counter.
(ii) Rate measurements. The rate of DNA synthesis was measured by incubating 0.3-ml duplicate samples of cells with 1.1 MuCi of [methyl-3H ]thymidine (New England Nuclear) for 4 min with vigorous agitation. After this time period, incorporation was stopped by adding 1.5 ml of cold trichloroacetic acid containing unlabeled thymidine (20 Mg/ml). The cells were collected on membrane filters (HA 0.45 pm, Millipore Corp.), washed with cold water containing
thymidine (20,Mg/ml), and dried, and the radioactivity was determined, as described above. (iii) Repair assay. To assay for extensive repair synthesis, cells were grown with [3H ]thymine for three generations to prelabel DNA. The 3H-label was removed by collecting cells on membrane filters, washing, and resuspending in fresh media, as described above. Unlabeled thymine (40 Mg/ml) was added to the fresh media to substantially dilute any radioactive breakdown products that might be reutilized. Samples were removed from the culture at intervals, and
the acid-precipitable radioactivity was assayed to detect any decrease in radioactivity, which would indicate repair. (iv) Density gradient analysis. A 35-ml culture of strain N-167 was grown for three generations in M9 light medium containing the usual required supplements and [14C jthymine (2 MCi/ml) at 30 C, and then transferred to 43 C for 75 to 100 min. The cells were then filtered, washed, and transferred to 35 ml of M9 heavy medium containing the usual required supplements and 5-bromouracil (Calbiochem) at 40 Mg/ml, "5NH4Cl at 0.2 mg/ml in place of "4NH4Cl, 0.1% ["3C]glucose, deuterated amino acids at 500 Mg/ml (Merck & Co.), and [3H]thymine at 4 MCi/ml. The culture was divided, and incubation at 30 C continued in the presence and absence of CAP (150 Mg/ml). Samples were removed at intervals, medium ice was added to arrest further growth, and the cells were collected by centrifugation. The cells were lysed by the addition of 1 ml of 0.02 M ethylenediaminetetraacetate and 1% sodium dodecyl sulfate. The lysate was then incubated either with trypsin or Pronase (Calbiochem) at 100 Mg/ml for 30 min at 37 C. The lysate was treated twice with phenol that had been saturated with 1% sodium dodecyl sulfate- 0.1 M tris(hydroxymethyl)aminomethane buffer (pH 9). The aqueous phase, containing the DNA, was then dialyzed against 0.1x SSC (0.15 M NaCl, plus 0.015 M sodium citrate). The DNA was then subjected to CsCl density gradient centrifugation for 43 to 45 h in a Beckman type 50 rotor at 40,000 rpm. Fractions were collected from the bottom of the tube, and the radioactivity in acid-precipitable counts was assayed as described above. RESULTS
When cultures of the DNA initiation mutants used in this study were shifted to 42 C, DNA synthesis ceased within 40 to 60 min as seen for strain PC-2 in Fig. 1A. After the culture was returned to 30 to 32 C, DNA synthesis began immediately and continued for several rounds. This result suggests that these mutants carry a reversible thermosensitive initiation defect and agrees with previous observations (1, 2, 4). Measurements of the rate of DNA synthesis after return to 30 to 32 C (Fig. 1B) showed that the rate increased for 10 min after the culture was returned to 32 C followed by a second increase occurring at about 20 to 30 min, followed by other increases at 40 to 60 min and
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PC-2
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60 to 80 min. It has been suggested that holding these mutants at nonpermissive temperature for a period of time sufficient to allow all rounds
to complete and cell division to occur, synchronizes rounds of replication upon return to permissive temperature (2). Also, the second increase has been shown to represent premature /initiation of a new growth fork, suggesting that M 42*C there was an accumulation at 42 C of some product(s) needed for initiation (1, 21). To determine whether all the products or B conditions needed to initiate more than one round of DNA synthesis had been produced at 42 C, cells were prelabeled for three generations, held for 100 min (1.5 to 2 generations) at 42 C, and retumed to 32 C in the presence of different inhibitors (CAP, PEA, or rifampin), which may block different steps needed to initiO 50 100 02260 ate synthesis (11, 13, 14, 24). The residual DNA MINUTES synthesis observed in the presence of these FIG. 1. S'ynthesis of DNA during and after incuba- inhibitors is shown in Fig. 2. Three of these tion at 42 C'.A culture of E. coli PC-2 was incubated treatments, 150 Asg of CAP per ml, 100 ,ug of for 100 min at 42 C before being returned to 30 C. (A) rifampin per ml, or 0.3% PEA, allowed an Incorporaticrn of [3H]thymine (2 MCi/4 Ag per ml) was amount of synthesis equal to about one DNA measured dluring growth at 42 and 30 C. (B) Rate of doubling. Low levels of CAP (30 ,ug/ml) also apDNA synth esis during growth at 42 C and 30 C was inevl by incubating monitored ait Intervals .t incu Atng0s.3-mi 0.3m samples sape peared to allow several rounds of DNA syntheof the cultu re with [3Hjthymidine (1.1 M.Ci/mI) for 4 sis to be initiated. These results may mean: (i) a protein sensitive to 30 Mg of CAP per ml may min.
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FIG. 2. Residual DNA synthesis in the presence of inhibitors after transfer to 32 C. Cultures of strain Hfr 165/120/6 and PC-2 were grown for three generations in [3Hjthymine (2 MCi/4 gg per ml) to uniformly label the chromosomes, incubated for 100 min at 42 C, and returned to 32 C in the presence of 30 (A) and 150 (0) Mg of CAP per ml, 0.3% PEA (0), 100 ug of rifampin per ml (A), or in the absence of inhibitors (0).
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have accumulated sufficiently at 42 C and, upon return to 32 C with 30,ug of CAP per ml present, more than one initiation event is permitted; or (ii) low levels of CAP cause much less inhibition of residual DNA synthesis, i.e., cause much less premature chain termination. During the course of this work, we found that the amount of CAP which allowed what appeared to be two rounds of replication in the residual synthesis experiments varied with the different strains. This result led us to test the effect of a spectrum of concentrations of CAP on the residual synthesis, as Cooper and Weustoff (6) have done. The results (Fig. 3) demonstrate that the amount of residual synthesis was inversely proportional to the CAP concentration and agree with the results of Cooper and Weustoff (6). As mentioned above, Lark (12) has suggested that this phenomenon is due to premature termination of DNA chain elongation which is proportional to the dose of CAP present. To examine inhibitor effects on initiation in this system in finer detail, therefore, the rate of DNA synthesis was monitored, using 4-min pulses of [3H ]thymidine given at various intervals after return to permissive temperature in the presence and absence of the inhibitors. Control cells began DNA synthesis immediately upon return to 32 C, and the rate of DNA synthesis doubled by 20 to 30 min, depending on the strain utilized (Fig. 4). In the presence of 150 Ag of CAP per ml, the initial rate of DNA synthesis at 0 to 10 min was greater than control in all strains. The rate then continued to
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increase for 40 to 50 min, as in strain 15T-ts489, or decreased and then remained constant until about 50 min followed by a decrease, as seen in strains Hfr 165/120/6 and N-167, or actually showed a decrease followed by an increase, as in strain PC-2. Since one round of DNA synthesis takes 40 min to complete (5), the continued substantial rate of synthesis after 40 min suggests that more than one round of DNA may be initiated, or that the rate of chain elongation is slowed so that the round of DNA synthesis which has initiated is taking longer to complete. The observation of a high rate of initial synthesis, coupled with the increase and decrease in rate observed in some strains, i.e., PC-2, is most consistent with the interpretation that more than one round of synthesis may be initiated in 150 sg of CAP per ml and that premature chain termination may explain the overall lower rate than the controls. In the presence of low levels of CAP (30 gg/ml), two increases in the rate of DNA synthesis at 0 and 30 to 35 min are clearly observed in all strains except strain 15T-ts489. Because the timing of the second round of replication observed in these strains in the presence of low levels of CAP is the same as the control, it is likely that this second rise in rate represents initiation rather than some possible repair synthesis due to degradation of the DNA by conditions caused by the presence of CAP. A further continuation of DNA synthesis is observed past 75 min when the round initiated at 35 min would be expected to be finished. This result may mean that, besides allowing a second initiation event, further initiations may occur in PC -2 the presence of 30 ,g of CAP per ml. An 12alternative possibility is that this synthesis represents slow completion of rounds of replication caused by a decreased rate of chain elongation. The effects of 100 Mg of rifampin per ml and 0.3% PEA on strains PC-2 and Hfr 165/120/6 have also been investigated. In the presence of these inhibitors (Fig. 5), again as seen above with CAP, the rate of DNA synthesis initially increased, decreased at about 20 min, and increased at 20 to 30 min in strain PC-2 and the other strains, suggesting that more than one initiation event can take place. In all cultures, 40 60 the overall rate of DNA synthesis was lower in pg/ml CAP FIG. 3. Effect of various concentrations of chlor- the presence of PEA or rifampin than in the amphenicol on residual DNA synthesis. Strain PC-2 control culture. This may be because these inhibitors either slow down the rate of DNA was labeled for three generations with [3HJthymine (2 MCi/4 Mig per ml). Varying concentrations of CAP were chain elongation or, perhaps, prevent some added, and the increase in radioactivity was followed segment of the cell population from recovering as a function of the CAP concentration. from the 42 C treatment.
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FIG. 4. Effect of high and low levels of CAP on the rate of DNA synthesis after 100 min at 42 C. Cultures of the initiation mutants were grown at 32 C and shifted to 42 C for 100 min. Upon return to 30 to 32 C permissive temperature, DNA synthesis was assayed at various time periods by incubating 0.3-ml portions of the culture with [3HJthymidine (1.1 uCi/ml) in the presence of 150 (A) or 30 jAg/ml (0) levels of CAP. Control culture was returned to 32 C without CAP (0).
Effects of amino acid starvation on DNA synthesis after growth at the restrictive temperature. To determine whether these results were due to some effect of the inhibitor per se or due to the inhibition of protein synthesis, the kinetics of DNA synthesis was observed for cells that were held at 42 C for 100 min and then returned to 32 C in the absence of amino acids. Except for possible protein turnover, the withdrawal of amino acids should be comparable to high levels of CAP, although more rounds of DNA synthesis may be able to finish according to Lark (12). The results (Fig. 6) show that the
pattern of DNA synthesis observed when amino acids are withheld is very similar to the pattern observed in the presence of high levels of CAP (see Fig. 4). Starvation for amino acids also resulted in an initial high rate of DNA synthesis greater than the control. This rate later decreased, and second increases were observed at 15 and 55 min, and perhaps a third increase at 75 min. Thus, the results obtained in the absence of amino acids appear similar to the effects of high CAP, indicating that the observed effects of high CAP on DNA synthesis at 32 C are probably caused by the inhibition of
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MINUTES MINUTES FIG. 5. Effect of rifampin and PEA on the rate of DNA synthesis after 100 min at 42 C. Cultures of strains Hfr 165/120/6 and PC-2 were grown at 32 C and shifted to 42 C for 100 min. Upon return to 32 C permissive temperature, DNA synthesis was assayed at various intervals (as described in Fig. 4) in the presence of 100 Ag of rifampin per ml (-), 3% PEA (A), or in the absence of inhibitors (0).
PC -2
MINUTES FIG. 6. Effect of amino acid starvation on the rate of DNA synthesis in strain PC-2 after 100 minutes at 42 C. Upon return to 32 C, DNA synthesis was assayed at various intervals (as described in Fig. 4) in the presence (0), or absence (A) of required amino acids. DNA incorporation was also measured in a control culture which was filtered, but to which amino acids were restored (-).
protein synthesis, rather than any direct effect of the inhibitor on DNA or the replication complex. Characterization of the DNA synthesis after return to permissive temperature. Since it is possible that increases in rate observed in the presence of the inhibitors might represent some type of synthesis other than the initiation of semiconservative replication, attempts were made to characterize the DNA synthesized after release of the cells to permisnew
sive conditions. If, for example, any of the treatments used in these experiments stimulated extensive repair synthesis, such repair should be detected as loss of acid-precipitable counts from [3H]thymine-prelabeled DNA (3, 23). To test for such repair, DNA was uniformly labeled by growth for several generations in [3H ]thymine and incubation for 100 min at 42 C. After this incubation, the cultures were filtered and returned to 32 C in the presence of excess unlabeled thymine plus rifampin, PEA, or high or low CAP. Although the results of the residual synthesis experiments in Fig. 2 show loss of acid-precipitable counts after prolonged exposure to 150 ,ug of CAP per ml, the results of the repair experiments described above (data not shown) indicated no appreciable loss of acid-precipitable counts within 100 min after return to 30 C in the presence of CAP (150 ,ug/ml or 30 ,g/ml), PEA or rifampin. To further characterize the DNA synthesized after return to permissive temperatures, density transfer experiments were attempted. Due to the large nucleotide pools observed in E. coli K-12 strains, and to the expansion of pools at 42 C, serious technical difficulties are encountered in experiments of this type because of the resulting long period of isotope mixing (9). Thus, to observe convincing density shifts, a culture of strain N-167 (which showed less pool problems than the other strains) was grown for three generations in light "C-labeled medium, and shifted to 43 C for 75 to 100 min in the same light medium. The cells were then filtered, washed, and transferred to medium containing [3C ]glucose, 15NH4Cl, deuterated amino acids, 5-bromouracil, and [3H]thymine. The culture
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was incubated at 32 C in the presence and absence of 150,ug of CAP per ml, and samples were taken at intervals and subjected to CsCl density gradient centrifugation. The results of this analysis can be seen in Fig. 7. The general pattem of DNA synthesis is the incorporation of the 3H-label into many peaks of increased density and the "4C-label (present in the light medium) appears to also be present in what probably represents twice replicated DNA of the highest density. These results are most likely due to the isotope mixing, mentioned above, which occurs for over 40 min. It is also evident that by 75 min not all of the prelabeled 14C has been replicated in the control culture, which is consistent with the autoradiographic finding that approximately 14% of the cells are unlabeled at 60 min after return to permissive temperature (data not shown). These probably represent cells in which some function was irreparably damaged by the treatment at nonpermissive temperature. Very little 'H-label is associated with the prelabeled light DNA, a result which would indicate, in conjunction with the test for extensive repair mentioned above, that probably little repair synthesis is taking place. A similar pattern of replication is seen in the cells exposed to CAP at 150 Ag/ml, except far less counts have been incorporated into DNA of increased densities, and far less prelabeled DNA has been replicated. However, the proportion of 3H counts located in DNA of the highest density is similar to that in the control. If the DNA of highest density does represent twice-replicated DNA, then it would appear that more than one initiation appears possible in the presence of 150 gg of CAP per ml, and the lower absolute incorporation is probably due to a lower rate of chain elongation or premature chain termination.
DISCUSSION The results reported here suggest that CAP, rifampin, and PEA may have multiple effects on the DNA synthetic rate. After holding a variety of reversible temperature-sensitive initiation mutants at nonpermissive temperature for over one generation time equivalent, and then releasing them to permissive temperature: (i) the above inhibitors do not appear to interfere with the first initiation event; (ii) in fact, the presence of 150 ,ug of CAP per ml appears to allow a higher initial rate of DNA synthesis than the control; and (iii) more than one initiation event appears possible, as suggested by rate experiments and density gradient analysis. If assumption iii is correct, then the apparent one round of synthesis allowed in the
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FIG. 7. Density gradient analysis of the DNA replicated after release to permissive temperature. Strain N-167 was grown in light medium containing ["C]thymine for three generations at 30 C and transferred to 43 C for 100 min. The cells were filtered, washed, transferred to heavy medium containing [3HJthymine, and incubated at 30 C in the presence and absence of 150 Ag of CAP per ml. Samples were taken at intervals and prepared for CsCI density gradient analysis. In all but frame C the 14C and 'H scales are the same: A, 14C; and 0, 3H.
presence of high CAP, reported by others (1, 2) and evidenced in the residual synthesis experiments in Fig. 2, may be due to a composite curve resulting from a combination of reinitiation events and slowing down of the rate and/or premature chain termination. A lower rate of chain elongation in the presence of CAP is evidenced by a lower amount of [3H]thymine
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incorporated into newly replicated DNA of increased densities, and a lower amount of light, prelabeled DNA which shows a density transfer as compared with the control at any given time. Also, for a one-min pulse, the highest class in the distribution of labeled cells, at 30 min after return to permissive conditions, is over 10 grains per cell in the control culture versus 1 to 2 grains per cell in the presence of 150 Ag of CAP per ml, whereas the distribution of unlabeled cells is very similar for both cultures at this time. By 60 min, however, 48% of the cells are unlabeled in the CAP culture as compared to 14% in the control (data not shown), which would indicate that CAP also causes premature chain termination. If the above interpretation of the data is correct, then it would appear that it is possible for cells to accumulate the capacity for more than one initiation event during the 100-min period at nonpermissive temperature. It is interesting to note here that we observe the same pattern of synthesis in rate experiments with 150 ug of CAP per ml for strain N-167 when held at nonpermissive temperature for only 45 min (data not shown). The mechanism whereby the cells gain this capacity, whether by accumulation of stable essential products actively required for initiation (7), or a product needed to remove a repressor (20), or by the dilution of a repressor (19) is not known. The fact that the initial rate is higher in the presence of 150 ,ug of CAP per ml suggests that more growing points are actively initiated when the synthesis of some protein(s) is prevented. If it is the absence of some protein that causes the increased rate, then the fact that cultures in which RNA synthesis is blocked with rifampin do not show an initial increase in rate may mean that the message for the protein(s) is made before the release to permissive conditions. Whether there are more than two initiation events that take place in the absence of further protein synthesis is not known. Also, it is difficult to draw any parallels with the prolonged DNA synthesis observed in the absence of protein synthesis, after periods of thymine starvation (8, 20), since DNA synthesis appears to essentially cease after 100 min in the absence of protein synthesis in the system which we have described. If the rate of chain elongation is slowed and prematurely terminated in the presence of CAP, then it follows that there may be some gene product that has a relatively short half-life and which is necessary for continued chain elongation. The results of the above experiments also
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suggest that great care should be taken in assessing the capacity of the cell to initiate based on the amount of incorporation achieved in the presence of various inhibitors of macromolecular synthesis in residual synthesis experiments. ACKNOWLEDGMENTS We thank Robert Posner for his technical assistance with these experiments. This work was supported by Public Health Research grant GM19189 from the National Institute of General Medical Sciences, and partially by contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project (Report no. UR-3490-620). LITERATURE CITED 1. Abe, M., and J. Tomizawa. 1971. Chromosome replication in an Escherichia coli K-12 mutant affected in the process of DNA replication. Genetics 69:1-15. 2. Beyersmann, D., M. Schlicht, and H. Schuster. 1971. Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K-12. Mol. Gen. Genet.
111:145-158. 3. Boyce, R. P., and P. Howard-Flanders. 1964. Release of ultraviolet light-induced thymine dimers from DNA in E. coliK-12. Proc. Nat. Acad. Sci. U.S.A. 51:293-300. 4. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 5. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. 6. Cooper, S., and G. Weusthoff. 1971. Comment on the use of chloramphenicol to study the initiation of deoxyribonucleic acid synthesis. J. Bacteriol. 106:709-711. 7. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 8. Kogama, T., and K. G. Lark. 1970. DNA replication in Escherichia coli: replication in absence of protein synthesis after replication inhibition. J. Mol. Biol. 52:143-164. 9. Kuempel, P. L. 1969. Temperature-sensitive initiation of chromosomal replication in a mutant of Escherichia coli. J. Bacteriol. 100:1302-1310. 10. Lark, C., and K. G. Lark. 1964. Evidence for two distinct aspects of the mechanism regulating chromosome replication in Escherichia coli. J. Mol. Biol. 10:120-136. 11. Lark, K. G. 1972. Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15T-. J. Mol. Biol. 64:47-60. 12. Lark, K. G. 1973. Initiation and termination of bacterial deoxyribonucleic acid replication in low concentrations of chloramphenicol. J. Bacteriol. 113:1066-1069. 13. Lark, K. G., and C. Lark. 1966. Regulation of chromosome replication in Escherichia coli: a comparison of the effects of phenethyl alcohol treatment with those of amino acid starvation. J. Mol. Biol. 20:9-19. 14. Lark, K. G., and H. Renger. 1969. Initiation of DNA replication in Escherichia coli 15T-; chronological dissection of three physiological processes required for initiation. J. Mol. Biol. 42:221-235. 15. Lark, K. G., T. Repko, and E. J. Hoffman. 1963. The effect of amino acid deprivation on subsequent DNA replication. Biochim. Biophys. Acta 76:9-24. 16. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis, p. 154-187. W. A. Benjamin, Inc., New York.
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17. Messer, W. 1972. Initiation of deoxyribonucleic acid replication of Escherichia coli B/r: chronology of events and transcriptional control of initiation. J. Bacteriol. 112:7-12. 18. Mitchison, J. M. 1971. The biology of the cell cycle, p. 167. Cambridge University Press, London. 19. Pritchard, R. H., P. T. Barth, and J. Collins. 1969. Control of DNA synthesis in bacteria. Symp. Soc. Gen. Microbiol. 19:263-279. 20. Rosenberg, B. H., L. B. Cavalieri, and G. Ungers. 1969. The negative control mechanism for E. coli DNA replication. Proc. Nat. Acad. Sci. U.S.A. 63:1410-1417. 21. Schubach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escherichia coli. J.
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Mol. Biol. 74:205-221. 22. Schwartz, M., and A. Worcel. 1971. Reinitiation of chromosome replication in a thermosensitive DNA mutant of Escherichia coli. II. Synchronization of chromosome replication after temperature shifts. J. Mol. Biol. 61:329-342. 23. Setlow, R. B., and W. L. Carrier. 1964. The disappearance of thymine dimers from DNA: an error correction mechanism. Proc. Nat. Acad. Sci. U.S.A. 51:226-231. 24. Ward, C. B., and D. Glaser. 1969. Analysis of the chloramphenicol-sensitive and chloramphenicol-resistant steps in the initiation of DNA synthesis in E. coli B/r. Proc. Nat. Acad. Sci. U.S.A. 64:905-912.
Vol. 121, No. 3
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Accumulation of the Capacity for Initiation of Deoxyribonucleic Acid Replication in Escherichia coli IRENE M. EVANS
AND
HELEN EBERLE*
Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642 Received for publication 15 October 1974
Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents. The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited. Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis. This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved. These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation.
In the bacterial cell cycle some proteins are synthesized continuously throughout the cycle while others are synthesized only periodically (14, 18, 24). Whether a protein is available for use continuously or only at particular times may have important consequences for control if such a protein is important in regulating cell cycle events, e.g., initiation of deoxyribonucleic acid (DNA) synthesis. Attempts have been made to determine whether the products necessary for DNA initiation are made continuously during a period when cells were held at a temperature nonpermissive for DNA initiation and synthesis. By measuring residual DNA synthesis in the presence of chloramphenicol (CAP) after temperature-sensitive initiation mutants had been held at nonpermissive temperature, Abe and Tomizawa (1) and Beyersmann et al. (2) found that the [3H]thymine counts in the culture approximately doubled, suggesting that only one cycle of DNA was able to be synthesized. Further protein synthesis at the permissive temperature seemed to be necessary to permit a second round of DNA synthesis. This confirmed the earlier findings of Schwartz and Worcel (22),who used a dnaB mutant to study this question. Schubach et al. (21), however, concluded from similar experiments that products needed for DNA synthesis were able to accumulate continuously at a nonpermissive temperature, allowing more than one round of DNA
synthesis without new protein synthesis upon return to permissive temperature. Recently, Lark (12) and Messer (17) obtained evidence which shows that CAP affects DNA chain elongation. Since most of the previous estimates of the number of rounds of DNA synthesized in the presence of CAP have been calculated from the amount of residual DNA synthesis in the presence of CAP (a calculation which assumed that CAP only blocked DNA initiation), it seemed possible that the number of initiation events might have been underestimated in these earlier studies. Therefore, the number of initiation events occurring in the presence of CAP and other initiation inhibitors has been reinvestigated by measuring the rate of DNA synthesis after the release df temperature-sensitive initiation mutants from nonpermissive conditions. The results indicate that more than one round of DNA synthesis may be initiated in the presence of these inhibitors. The failure to detect such reinitiation events in previous residual synthesis experiments may be explained by the possible masking of reinitiation by premature chain termination. MATERIALS AND METHODS Bacteria. The following strains were utilized: Escherichia coli 15T-ts-489, dnaC (Thy-, Arg-, Met-, Trp-; obtained from K. G. Lark); E. coli K-12, dna252A (2) (Thi-, Thy-, Pro-, StrS; obtained from F. Bonhoeffer and called Hfr 165/120/6); E. coli K-12,
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dna2C (Thy-, Leu-, StrR; obtained from P. Carl and called PC-2) (4); E. coli K-12, dnaA (Thy-, Thi-; obtained from Tomizawa and called N-167) (1). Growth conditions. All cells were grown in M9 minimal medium (15) to which required supplements were added as needed (micrograms per milliliter): thymine, 4; proline, 20; arginine, 20; methionine, 20; tryptophan, 20; leucine, 40; and thiamine hydrochloride (vitamin B1), 2. The K-12 strains were normally grown with the addition of vitamin-free Casamino Acids (1%) (Difco). Medium changes were accomplished by collecting cells on 0.45-Mm membrane filters (Millipore Corp. or Schleicher and Schuell), washing with prewarmed M9 medium, and suspending cells in the desired medium. The doubling time for the strains Hfr 165/120/6, PC-2, and N-167 in minimal M9 media was 55 to 60 min at 30 to 32 C. The generation time for strain 15T-ts-489, grown at 30 to 32 C with the required supplements, was 45 min. In all experiments, 30 to 32 C was the permissive temperature for DNA initiation which ceased when the strains were transferred to 42 C (43 C for strain N-167). Inhibitors. All inhibitors blocked DNA synthesis within 40 to 80 min after addition. CAP (Parke, Davis and Co.), at 150 Mg/ml, inhibited protein synthesis essentially immediately, whereas 30,Mg of CAP per ml reduced protein synthesis to a level 10 to 30% that of the control. Phenethyl alcohol (PEA; Eastman Kodak Co.), at 0.3%, allowed a low level of ribonucleic acid synthesis and permitted protein synthesis at a level 20 to 40% that of the control. Rifampin (Calbiochem), at 100 Mg/ml, blocked ribonucleic acid and protein synthesis essentially immediately upon addition. CAP stock solutions were prepared fresh daily in a 47.5% ethanol solution at a concentration of 3 to 6 mg/ml. A rifampin stock solution was made by dissolving 10 mg of rifampin in methanol, and stored in the dark at 0 C. PEA was stored at room temperature and added to cultures at the necessary concentrations. Control experiments demonstrated that neither methanol nor ethanol had any effect on the amount of radioactivity incorporated into DNA under the experimental conditions at 30 to 32 C. Radioactive labeling: (i) Continuous label experiments. For experiments in which DNA synthesis was measured, [methyl-3H ]thymine (New England Nuclear) at 2 MCi/4 Mg per ml was added. Duplicate 0.1-ml samples of culture were removed and precipitated by 1 ml of 5%' cold trichloroacetic acid. Precipitates were collected on Reeve Angel 934AH glass-fiber filters (Clifton, N.J.), washed with cold distilled water, and dried, and the radioactivity was determined on a Beckman model LS250 scintillation counter.
(ii) Rate measurements. The rate of DNA synthesis was measured by incubating 0.3-ml duplicate samples of cells with 1.1 MuCi of [methyl-3H ]thymidine (New England Nuclear) for 4 min with vigorous agitation. After this time period, incorporation was stopped by adding 1.5 ml of cold trichloroacetic acid containing unlabeled thymidine (20 Mg/ml). The cells were collected on membrane filters (HA 0.45 pm, Millipore Corp.), washed with cold water containing
thymidine (20,Mg/ml), and dried, and the radioactivity was determined, as described above. (iii) Repair assay. To assay for extensive repair synthesis, cells were grown with [3H ]thymine for three generations to prelabel DNA. The 3H-label was removed by collecting cells on membrane filters, washing, and resuspending in fresh media, as described above. Unlabeled thymine (40 Mg/ml) was added to the fresh media to substantially dilute any radioactive breakdown products that might be reutilized. Samples were removed from the culture at intervals, and
the acid-precipitable radioactivity was assayed to detect any decrease in radioactivity, which would indicate repair. (iv) Density gradient analysis. A 35-ml culture of strain N-167 was grown for three generations in M9 light medium containing the usual required supplements and [14C jthymine (2 MCi/ml) at 30 C, and then transferred to 43 C for 75 to 100 min. The cells were then filtered, washed, and transferred to 35 ml of M9 heavy medium containing the usual required supplements and 5-bromouracil (Calbiochem) at 40 Mg/ml, "5NH4Cl at 0.2 mg/ml in place of "4NH4Cl, 0.1% ["3C]glucose, deuterated amino acids at 500 Mg/ml (Merck & Co.), and [3H]thymine at 4 MCi/ml. The culture was divided, and incubation at 30 C continued in the presence and absence of CAP (150 Mg/ml). Samples were removed at intervals, medium ice was added to arrest further growth, and the cells were collected by centrifugation. The cells were lysed by the addition of 1 ml of 0.02 M ethylenediaminetetraacetate and 1% sodium dodecyl sulfate. The lysate was then incubated either with trypsin or Pronase (Calbiochem) at 100 Mg/ml for 30 min at 37 C. The lysate was treated twice with phenol that had been saturated with 1% sodium dodecyl sulfate- 0.1 M tris(hydroxymethyl)aminomethane buffer (pH 9). The aqueous phase, containing the DNA, was then dialyzed against 0.1x SSC (0.15 M NaCl, plus 0.015 M sodium citrate). The DNA was then subjected to CsCl density gradient centrifugation for 43 to 45 h in a Beckman type 50 rotor at 40,000 rpm. Fractions were collected from the bottom of the tube, and the radioactivity in acid-precipitable counts was assayed as described above. RESULTS
When cultures of the DNA initiation mutants used in this study were shifted to 42 C, DNA synthesis ceased within 40 to 60 min as seen for strain PC-2 in Fig. 1A. After the culture was returned to 30 to 32 C, DNA synthesis began immediately and continued for several rounds. This result suggests that these mutants carry a reversible thermosensitive initiation defect and agrees with previous observations (1, 2, 4). Measurements of the rate of DNA synthesis after return to 30 to 32 C (Fig. 1B) showed that the rate increased for 10 min after the culture was returned to 32 C followed by a second increase occurring at about 20 to 30 min, followed by other increases at 40 to 60 min and
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60 to 80 min. It has been suggested that holding these mutants at nonpermissive temperature for a period of time sufficient to allow all rounds
to complete and cell division to occur, synchronizes rounds of replication upon return to permissive temperature (2). Also, the second increase has been shown to represent premature /initiation of a new growth fork, suggesting that M 42*C there was an accumulation at 42 C of some product(s) needed for initiation (1, 21). To determine whether all the products or B conditions needed to initiate more than one round of DNA synthesis had been produced at 42 C, cells were prelabeled for three generations, held for 100 min (1.5 to 2 generations) at 42 C, and retumed to 32 C in the presence of different inhibitors (CAP, PEA, or rifampin), which may block different steps needed to initiO 50 100 02260 ate synthesis (11, 13, 14, 24). The residual DNA MINUTES synthesis observed in the presence of these FIG. 1. S'ynthesis of DNA during and after incuba- inhibitors is shown in Fig. 2. Three of these tion at 42 C'.A culture of E. coli PC-2 was incubated treatments, 150 Asg of CAP per ml, 100 ,ug of for 100 min at 42 C before being returned to 30 C. (A) rifampin per ml, or 0.3% PEA, allowed an Incorporaticrn of [3H]thymine (2 MCi/4 Ag per ml) was amount of synthesis equal to about one DNA measured dluring growth at 42 and 30 C. (B) Rate of doubling. Low levels of CAP (30 ,ug/ml) also apDNA synth esis during growth at 42 C and 30 C was inevl by incubating monitored ait Intervals .t incu Atng0s.3-mi 0.3m samples sape peared to allow several rounds of DNA syntheof the cultu re with [3Hjthymidine (1.1 M.Ci/mI) for 4 sis to be initiated. These results may mean: (i) a protein sensitive to 30 Mg of CAP per ml may min.
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FIG. 2. Residual DNA synthesis in the presence of inhibitors after transfer to 32 C. Cultures of strain Hfr 165/120/6 and PC-2 were grown for three generations in [3Hjthymine (2 MCi/4 gg per ml) to uniformly label the chromosomes, incubated for 100 min at 42 C, and returned to 32 C in the presence of 30 (A) and 150 (0) Mg of CAP per ml, 0.3% PEA (0), 100 ug of rifampin per ml (A), or in the absence of inhibitors (0).
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have accumulated sufficiently at 42 C and, upon return to 32 C with 30,ug of CAP per ml present, more than one initiation event is permitted; or (ii) low levels of CAP cause much less inhibition of residual DNA synthesis, i.e., cause much less premature chain termination. During the course of this work, we found that the amount of CAP which allowed what appeared to be two rounds of replication in the residual synthesis experiments varied with the different strains. This result led us to test the effect of a spectrum of concentrations of CAP on the residual synthesis, as Cooper and Weustoff (6) have done. The results (Fig. 3) demonstrate that the amount of residual synthesis was inversely proportional to the CAP concentration and agree with the results of Cooper and Weustoff (6). As mentioned above, Lark (12) has suggested that this phenomenon is due to premature termination of DNA chain elongation which is proportional to the dose of CAP present. To examine inhibitor effects on initiation in this system in finer detail, therefore, the rate of DNA synthesis was monitored, using 4-min pulses of [3H ]thymidine given at various intervals after return to permissive temperature in the presence and absence of the inhibitors. Control cells began DNA synthesis immediately upon return to 32 C, and the rate of DNA synthesis doubled by 20 to 30 min, depending on the strain utilized (Fig. 4). In the presence of 150 Ag of CAP per ml, the initial rate of DNA synthesis at 0 to 10 min was greater than control in all strains. The rate then continued to
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increase for 40 to 50 min, as in strain 15T-ts489, or decreased and then remained constant until about 50 min followed by a decrease, as seen in strains Hfr 165/120/6 and N-167, or actually showed a decrease followed by an increase, as in strain PC-2. Since one round of DNA synthesis takes 40 min to complete (5), the continued substantial rate of synthesis after 40 min suggests that more than one round of DNA may be initiated, or that the rate of chain elongation is slowed so that the round of DNA synthesis which has initiated is taking longer to complete. The observation of a high rate of initial synthesis, coupled with the increase and decrease in rate observed in some strains, i.e., PC-2, is most consistent with the interpretation that more than one round of synthesis may be initiated in 150 sg of CAP per ml and that premature chain termination may explain the overall lower rate than the controls. In the presence of low levels of CAP (30 gg/ml), two increases in the rate of DNA synthesis at 0 and 30 to 35 min are clearly observed in all strains except strain 15T-ts489. Because the timing of the second round of replication observed in these strains in the presence of low levels of CAP is the same as the control, it is likely that this second rise in rate represents initiation rather than some possible repair synthesis due to degradation of the DNA by conditions caused by the presence of CAP. A further continuation of DNA synthesis is observed past 75 min when the round initiated at 35 min would be expected to be finished. This result may mean that, besides allowing a second initiation event, further initiations may occur in PC -2 the presence of 30 ,g of CAP per ml. An 12alternative possibility is that this synthesis represents slow completion of rounds of replication caused by a decreased rate of chain elongation. The effects of 100 Mg of rifampin per ml and 0.3% PEA on strains PC-2 and Hfr 165/120/6 have also been investigated. In the presence of these inhibitors (Fig. 5), again as seen above with CAP, the rate of DNA synthesis initially increased, decreased at about 20 min, and increased at 20 to 30 min in strain PC-2 and the other strains, suggesting that more than one initiation event can take place. In all cultures, 40 60 the overall rate of DNA synthesis was lower in pg/ml CAP FIG. 3. Effect of various concentrations of chlor- the presence of PEA or rifampin than in the amphenicol on residual DNA synthesis. Strain PC-2 control culture. This may be because these inhibitors either slow down the rate of DNA was labeled for three generations with [3HJthymine (2 MCi/4 Mig per ml). Varying concentrations of CAP were chain elongation or, perhaps, prevent some added, and the increase in radioactivity was followed segment of the cell population from recovering as a function of the CAP concentration. from the 42 C treatment.
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FIG. 4. Effect of high and low levels of CAP on the rate of DNA synthesis after 100 min at 42 C. Cultures of the initiation mutants were grown at 32 C and shifted to 42 C for 100 min. Upon return to 30 to 32 C permissive temperature, DNA synthesis was assayed at various time periods by incubating 0.3-ml portions of the culture with [3HJthymidine (1.1 uCi/ml) in the presence of 150 (A) or 30 jAg/ml (0) levels of CAP. Control culture was returned to 32 C without CAP (0).
Effects of amino acid starvation on DNA synthesis after growth at the restrictive temperature. To determine whether these results were due to some effect of the inhibitor per se or due to the inhibition of protein synthesis, the kinetics of DNA synthesis was observed for cells that were held at 42 C for 100 min and then returned to 32 C in the absence of amino acids. Except for possible protein turnover, the withdrawal of amino acids should be comparable to high levels of CAP, although more rounds of DNA synthesis may be able to finish according to Lark (12). The results (Fig. 6) show that the
pattern of DNA synthesis observed when amino acids are withheld is very similar to the pattern observed in the presence of high levels of CAP (see Fig. 4). Starvation for amino acids also resulted in an initial high rate of DNA synthesis greater than the control. This rate later decreased, and second increases were observed at 15 and 55 min, and perhaps a third increase at 75 min. Thus, the results obtained in the absence of amino acids appear similar to the effects of high CAP, indicating that the observed effects of high CAP on DNA synthesis at 32 C are probably caused by the inhibition of
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MINUTES MINUTES FIG. 5. Effect of rifampin and PEA on the rate of DNA synthesis after 100 min at 42 C. Cultures of strains Hfr 165/120/6 and PC-2 were grown at 32 C and shifted to 42 C for 100 min. Upon return to 32 C permissive temperature, DNA synthesis was assayed at various intervals (as described in Fig. 4) in the presence of 100 Ag of rifampin per ml (-), 3% PEA (A), or in the absence of inhibitors (0).
PC -2
MINUTES FIG. 6. Effect of amino acid starvation on the rate of DNA synthesis in strain PC-2 after 100 minutes at 42 C. Upon return to 32 C, DNA synthesis was assayed at various intervals (as described in Fig. 4) in the presence (0), or absence (A) of required amino acids. DNA incorporation was also measured in a control culture which was filtered, but to which amino acids were restored (-).
protein synthesis, rather than any direct effect of the inhibitor on DNA or the replication complex. Characterization of the DNA synthesis after return to permissive temperature. Since it is possible that increases in rate observed in the presence of the inhibitors might represent some type of synthesis other than the initiation of semiconservative replication, attempts were made to characterize the DNA synthesized after release of the cells to permisnew
sive conditions. If, for example, any of the treatments used in these experiments stimulated extensive repair synthesis, such repair should be detected as loss of acid-precipitable counts from [3H]thymine-prelabeled DNA (3, 23). To test for such repair, DNA was uniformly labeled by growth for several generations in [3H ]thymine and incubation for 100 min at 42 C. After this incubation, the cultures were filtered and returned to 32 C in the presence of excess unlabeled thymine plus rifampin, PEA, or high or low CAP. Although the results of the residual synthesis experiments in Fig. 2 show loss of acid-precipitable counts after prolonged exposure to 150 ,ug of CAP per ml, the results of the repair experiments described above (data not shown) indicated no appreciable loss of acid-precipitable counts within 100 min after return to 30 C in the presence of CAP (150 ,ug/ml or 30 ,g/ml), PEA or rifampin. To further characterize the DNA synthesized after return to permissive temperatures, density transfer experiments were attempted. Due to the large nucleotide pools observed in E. coli K-12 strains, and to the expansion of pools at 42 C, serious technical difficulties are encountered in experiments of this type because of the resulting long period of isotope mixing (9). Thus, to observe convincing density shifts, a culture of strain N-167 (which showed less pool problems than the other strains) was grown for three generations in light "C-labeled medium, and shifted to 43 C for 75 to 100 min in the same light medium. The cells were then filtered, washed, and transferred to medium containing [3C ]glucose, 15NH4Cl, deuterated amino acids, 5-bromouracil, and [3H]thymine. The culture
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was incubated at 32 C in the presence and absence of 150,ug of CAP per ml, and samples were taken at intervals and subjected to CsCl density gradient centrifugation. The results of this analysis can be seen in Fig. 7. The general pattem of DNA synthesis is the incorporation of the 3H-label into many peaks of increased density and the "4C-label (present in the light medium) appears to also be present in what probably represents twice replicated DNA of the highest density. These results are most likely due to the isotope mixing, mentioned above, which occurs for over 40 min. It is also evident that by 75 min not all of the prelabeled 14C has been replicated in the control culture, which is consistent with the autoradiographic finding that approximately 14% of the cells are unlabeled at 60 min after return to permissive temperature (data not shown). These probably represent cells in which some function was irreparably damaged by the treatment at nonpermissive temperature. Very little 'H-label is associated with the prelabeled light DNA, a result which would indicate, in conjunction with the test for extensive repair mentioned above, that probably little repair synthesis is taking place. A similar pattern of replication is seen in the cells exposed to CAP at 150 Ag/ml, except far less counts have been incorporated into DNA of increased densities, and far less prelabeled DNA has been replicated. However, the proportion of 3H counts located in DNA of the highest density is similar to that in the control. If the DNA of highest density does represent twice-replicated DNA, then it would appear that more than one initiation appears possible in the presence of 150 gg of CAP per ml, and the lower absolute incorporation is probably due to a lower rate of chain elongation or premature chain termination.
DISCUSSION The results reported here suggest that CAP, rifampin, and PEA may have multiple effects on the DNA synthetic rate. After holding a variety of reversible temperature-sensitive initiation mutants at nonpermissive temperature for over one generation time equivalent, and then releasing them to permissive temperature: (i) the above inhibitors do not appear to interfere with the first initiation event; (ii) in fact, the presence of 150 ,ug of CAP per ml appears to allow a higher initial rate of DNA synthesis than the control; and (iii) more than one initiation event appears possible, as suggested by rate experiments and density gradient analysis. If assumption iii is correct, then the apparent one round of synthesis allowed in the
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FIG. 7. Density gradient analysis of the DNA replicated after release to permissive temperature. Strain N-167 was grown in light medium containing ["C]thymine for three generations at 30 C and transferred to 43 C for 100 min. The cells were filtered, washed, transferred to heavy medium containing [3HJthymine, and incubated at 30 C in the presence and absence of 150 Ag of CAP per ml. Samples were taken at intervals and prepared for CsCI density gradient analysis. In all but frame C the 14C and 'H scales are the same: A, 14C; and 0, 3H.
presence of high CAP, reported by others (1, 2) and evidenced in the residual synthesis experiments in Fig. 2, may be due to a composite curve resulting from a combination of reinitiation events and slowing down of the rate and/or premature chain termination. A lower rate of chain elongation in the presence of CAP is evidenced by a lower amount of [3H]thymine
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incorporated into newly replicated DNA of increased densities, and a lower amount of light, prelabeled DNA which shows a density transfer as compared with the control at any given time. Also, for a one-min pulse, the highest class in the distribution of labeled cells, at 30 min after return to permissive conditions, is over 10 grains per cell in the control culture versus 1 to 2 grains per cell in the presence of 150 Ag of CAP per ml, whereas the distribution of unlabeled cells is very similar for both cultures at this time. By 60 min, however, 48% of the cells are unlabeled in the CAP culture as compared to 14% in the control (data not shown), which would indicate that CAP also causes premature chain termination. If the above interpretation of the data is correct, then it would appear that it is possible for cells to accumulate the capacity for more than one initiation event during the 100-min period at nonpermissive temperature. It is interesting to note here that we observe the same pattern of synthesis in rate experiments with 150 ug of CAP per ml for strain N-167 when held at nonpermissive temperature for only 45 min (data not shown). The mechanism whereby the cells gain this capacity, whether by accumulation of stable essential products actively required for initiation (7), or a product needed to remove a repressor (20), or by the dilution of a repressor (19) is not known. The fact that the initial rate is higher in the presence of 150 ,ug of CAP per ml suggests that more growing points are actively initiated when the synthesis of some protein(s) is prevented. If it is the absence of some protein that causes the increased rate, then the fact that cultures in which RNA synthesis is blocked with rifampin do not show an initial increase in rate may mean that the message for the protein(s) is made before the release to permissive conditions. Whether there are more than two initiation events that take place in the absence of further protein synthesis is not known. Also, it is difficult to draw any parallels with the prolonged DNA synthesis observed in the absence of protein synthesis, after periods of thymine starvation (8, 20), since DNA synthesis appears to essentially cease after 100 min in the absence of protein synthesis in the system which we have described. If the rate of chain elongation is slowed and prematurely terminated in the presence of CAP, then it follows that there may be some gene product that has a relatively short half-life and which is necessary for continued chain elongation. The results of the above experiments also
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suggest that great care should be taken in assessing the capacity of the cell to initiate based on the amount of incorporation achieved in the presence of various inhibitors of macromolecular synthesis in residual synthesis experiments. ACKNOWLEDGMENTS We thank Robert Posner for his technical assistance with these experiments. This work was supported by Public Health Research grant GM19189 from the National Institute of General Medical Sciences, and partially by contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project (Report no. UR-3490-620). LITERATURE CITED 1. Abe, M., and J. Tomizawa. 1971. Chromosome replication in an Escherichia coli K-12 mutant affected in the process of DNA replication. Genetics 69:1-15. 2. Beyersmann, D., M. Schlicht, and H. Schuster. 1971. Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K-12. Mol. Gen. Genet.
111:145-158. 3. Boyce, R. P., and P. Howard-Flanders. 1964. Release of ultraviolet light-induced thymine dimers from DNA in E. coliK-12. Proc. Nat. Acad. Sci. U.S.A. 51:293-300. 4. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122. 5. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. 6. Cooper, S., and G. Weusthoff. 1971. Comment on the use of chloramphenicol to study the initiation of deoxyribonucleic acid synthesis. J. Bacteriol. 106:709-711. 7. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 8. Kogama, T., and K. G. Lark. 1970. DNA replication in Escherichia coli: replication in absence of protein synthesis after replication inhibition. J. Mol. Biol. 52:143-164. 9. Kuempel, P. L. 1969. Temperature-sensitive initiation of chromosomal replication in a mutant of Escherichia coli. J. Bacteriol. 100:1302-1310. 10. Lark, C., and K. G. Lark. 1964. Evidence for two distinct aspects of the mechanism regulating chromosome replication in Escherichia coli. J. Mol. Biol. 10:120-136. 11. Lark, K. G. 1972. Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15T-. J. Mol. Biol. 64:47-60. 12. Lark, K. G. 1973. Initiation and termination of bacterial deoxyribonucleic acid replication in low concentrations of chloramphenicol. J. Bacteriol. 113:1066-1069. 13. Lark, K. G., and C. Lark. 1966. Regulation of chromosome replication in Escherichia coli: a comparison of the effects of phenethyl alcohol treatment with those of amino acid starvation. J. Mol. Biol. 20:9-19. 14. Lark, K. G., and H. Renger. 1969. Initiation of DNA replication in Escherichia coli 15T-; chronological dissection of three physiological processes required for initiation. J. Mol. Biol. 42:221-235. 15. Lark, K. G., T. Repko, and E. J. Hoffman. 1963. The effect of amino acid deprivation on subsequent DNA replication. Biochim. Biophys. Acta 76:9-24. 16. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis, p. 154-187. W. A. Benjamin, Inc., New York.
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17. Messer, W. 1972. Initiation of deoxyribonucleic acid replication of Escherichia coli B/r: chronology of events and transcriptional control of initiation. J. Bacteriol. 112:7-12. 18. Mitchison, J. M. 1971. The biology of the cell cycle, p. 167. Cambridge University Press, London. 19. Pritchard, R. H., P. T. Barth, and J. Collins. 1969. Control of DNA synthesis in bacteria. Symp. Soc. Gen. Microbiol. 19:263-279. 20. Rosenberg, B. H., L. B. Cavalieri, and G. Ungers. 1969. The negative control mechanism for E. coli DNA replication. Proc. Nat. Acad. Sci. U.S.A. 63:1410-1417. 21. Schubach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escherichia coli. J.
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Mol. Biol. 74:205-221. 22. Schwartz, M., and A. Worcel. 1971. Reinitiation of chromosome replication in a thermosensitive DNA mutant of Escherichia coli. II. Synchronization of chromosome replication after temperature shifts. J. Mol. Biol. 61:329-342. 23. Setlow, R. B., and W. L. Carrier. 1964. The disappearance of thymine dimers from DNA: an error correction mechanism. Proc. Nat. Acad. Sci. U.S.A. 51:226-231. 24. Ward, C. B., and D. Glaser. 1969. Analysis of the chloramphenicol-sensitive and chloramphenicol-resistant steps in the initiation of DNA synthesis in E. coli B/r. Proc. Nat. Acad. Sci. U.S.A. 64:905-912.
