Inhibition of Replication Gap Closure in Escherichia coli by Near ...

JOURNAL OF BACTERIOLOGY, JUIY 1974, p. 62-69 Copyright ( 1974 American Society for Microbiology

Vol. 119, No. 1 Printed in U.S.A.

Inhibition of Replication Gap Closure in Escherichia coli by Near-Ultraviolet Light Photoproducts of L-Tryptophan G. YOAKUM, W. FERRON,1 A. EISENSTARK,2 AND R. B. WEBB Divisions of Biological and Medical Research and of Biological Sciences, Argonne National Laboratory, Argonne, Illinois 60439

Received for publication 22 February 1974

Near-ultraviolet photoproducts of L-tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polAl, and recA recB double mutants of Escherichia coli. Wild-type cells labeled in their DNA with [3H]thymidine in the presence of TP produced small pieces of DNA (7 x 106 daltons), which corresponded in size to late replicative intermediates of discontinuous DNA synthesis. Moreover, when TP was present, it took five times longer to chase the low-molecular-weight DNA pieces into highmolecular-weight DNA. The observation of replicative intermediates in the presence of TP, and their slow chase into high-molecular-weight DNA in the presence of TP, is strong evidence that TP stabilizes replication gaps in E. coli DNA. Although TP slowed DNA replication in wild-type cells, this effect was transient and DNA synthesis eventually resumed at a normal rate. However, in polAl and recA recB mutants, DNA synthesis was completely inhibited. Determinations of size and total counts of cells incubated in TP suggested that TP uncouples cell division from DNA replication in recA recB mutants, whereas these processes remain coupled in wild-type cells and in uvrA and polAl mutants. The slow chase of TP-stabilized pieces of DNA in the presence of TP suggested that the selective effect of TP on DNA synthesis and viability in repair-deficient mutants is a result of TP inhibition of replication gap closure. It has been postulated (18) that semiconservative replication of deoxyribonucleic acid (DNA) involves the discontinuous synthesis of short DNA pieces that are early intermediates in chromosome formation. The generation of nascent DNA fragments observed by Okazaki and others (19) may be a multi-step process involving the unwinding of DNA, synthesis of a ribonucleic acid (RNA) primer, and subsequent 5'-3' DNA synthesis using the 3-OH of the RNA primer as a starting point (20). This discontinuous mode of DNA replication produces nascent DNA pieces separated by a gap and the RNA primer (18, 20). Thus, completion of replication may require nucleolytic removal of the RNA, repair synthesis of the gap, and ligase joining of the nascent pieces. Therefore, the inhibition of replication gap closing by near-ultraviolet photoproducts of L-tryptophan (hereafter referred to as TP) could result from inhibition at one or more of these steps. We have previously reported that rec mutants are especially sensitive to TP, whereas uvr mutants are no more sensitive to TP than are

wild-type cells (22). The observation that the viability of uvr or polAl mutants was not reduced by exposure to doses of TP that killed rec or exr cells suggested that TP might block some part of the excision repair system. We found that TP sensitizes both wild-type and excision repair-defective cells to ultraviolet light and X-ray killing when present during irradiation, and that TP inhibits the repair of X ray-induced single-strand breaks in Escherichia coli (G. Yoakum, A. Eisenstark, and R. B. Webb, manuscript in preparation). In this paper, we report that TP has a differential effect on DNA synthesis and cell division in wild-type cells, uvr and polAl mutants and a recA recB double mutant. We also present evidence suggesting that the differential effects of TP on DNA synthesis, cell division, and viability of wild-type and repair-deficient mutants may result from the effect of TP inhibition of replication gap closing on the recombination repair system of these strains. MATERIALS AND METHODS Bacterial strains. The following strains were used in the experiments described below: W3110 wild type, thy; AB2500 uvrA thy; P3478 polAl, thy; and X9247 recA recB thy. All strains used were derived from E.

Present address: Missouri Southern State College, Joplin, Mo. 64801. 2Present address: 105 Tucker Hall, University of MissouriColumbia, Columbia, Mo. 65201. '

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coli K-12. The genotypes and phenotypes are listed in Table 1. Growth and media. Log-phase cells were obtained for labeling and growth experiments by a 1: 20 dilution of ovemight cultures that had grown through at least 20 generations of log-phase growth and had not exceeded 2 x 108 cells/ml. Minimal medium was buffered with M9 salts supplemented as follows (in micrograms per milliliter): glucose, 400; L-tryptophan, 20; L-proline, 10; L-histidine, 10; Casamino Acids (vitamin free), 200; niacin, 1; thiamine, 1; and thymine, 10. The M9 salts contained the following (in grams per liter): NH4Cl, 1.0; Na2HPO4.7H20, 6.0; KH2PO4, 3.0; MgSO4 7H20, 0.2; and NaCl, 0.5. This medium was used for growth in all experiments and will hereafter be referred to as M9 medium. The cells were incubated in a 37 C constant-temperature room, and all experimental manipulations were carried out in this room with prewarmed material and media. Photoproduct preparation. Tryptophan photoproduct was prepared by aerobic irradiation of a 5 mg/ml aqueous solution of L-tryptophan (pH 8.2) to

measure DNA synthesis, 20-,uliter samples were placed on Whatman 542 filter paper disks saturated with 5% trichloroacetic acid. The samples were then washed twice with 5% trichloroacetic acid and once with 95% ethanol in an ice bath to remove unincorporated trichloroacetic acid-soluble counts. The filter paper disks were dried and placed in scintillation vials with about 5 ml of Packard Permablend 1 (5.5 g/liter) in toluene and counted in a Beckman 100 liquid scintillation counter. Standards were prepared on Whatman 542 filters and counted as above to determine the H: 14C spillover ratios, and each sample was normalized. The response of cell division to TP was determined by removing 0.1 ml of cells at the same time samples were taken for DNA synthesis determinations and placing the cells in 10 ml of 0.1 N HCl for counting and sizing with a modified Coulter counter. This cell counter is a modification of the standard counter, with the aperture and electronics selected for accurate counting and sizing of bacteria (11). Analysis of DNA. The size of prelabeled DNA and of DNA synthesized in the presence of TP was an approximate absorbed dose of 107 ergs/mm2 of determined by sedimentation through alkaline su290-nm light from a Hanovia 2.5-kW xenon mercury crose gradients by a modification of the procedure of arc lamp and a Bausch & Lomb 500-mm monoMcGrath and Williams (14). Log-phase W3110 cells chromator. The monochromatic light included some were prelabeled in M9 medium with 1.0 MCi long-wavelength scattered light and an approximate of [14C]thymine per ml (45 mCi/mmol). The preladispersion band of 280 to 300 nm. This light source beled cells were harvested at 2 x 108 cells/ml on a was chosen for use after an action spectrum study membrane filter and resuspended in TP-M9 medium indicating that 290 nm is the most effective wave- with 25 MCi of [3H]thymidine per ml (1.0 Ci/mmol). length for the production of tryptophan photoproduct The cells were allowed to incorporate [3Hlthymidine (G. Yoakum, M.S. thesis, Kansas State University, for 15 min at 37 C in the presence of TP and then were 1971). However, it is also possible to make tryptophan filtered and resuspended in TP-M9 or in M9 medium photoproduct with a broad-spectrum black-light (22). substituted with 100 Mg of cold thymidine per ml. A The photoproduct was diluted into M9 medium 0.1-ml sample was removed and placed on ice (hereafter referred to as TP-M9) to a final concentra- with KCN tris(hydroxymethyl)aminomethane ethyl tion equivalent to 375 Mg of tryptophan per ml in the enediaminetetraacetic acid stop mix (16) at time unirradiated solution for all cell incubations. zero and various times for 5 min. To avoid breakage DNA synthesis and cell division. Log-phase cells of the DNA, the cells were lysed on top of a 5 to 20% were prelabeled for three to five generations in M9 alkaline sucrose gradient by first layering 0.2 ml of medium with 1.0 MCi of [14C ]thymine per ml (specific 0.05% sodium dodecyl sulfate in a solution of 0.5 N activity 45 mCi/mmol). The prelabeled cells were NaOH, to which 50 Mlliters of cells (about 107) was then harvested on membrane filters (washed, 0.22 Mm added. After allowing 30 min for lysis at room tempore size; Millipore Corp.) and resuspended in M9 perature, the gradients were centrifuged by using medium or TP-M9 medium with 10 MCi of [8H]thymi- an SW56Ti rotor in a Beckman L-2 65B ultracentridine per ml (specific activity, 1.5 Ci/mmol). To fuge at 35,000 rpm for 95 min at 20 C. Eight-drop -

-

-

TABLE 1. List of Escherichia coli strains described in the text Ultraviolet light

TP responsec

Strain no.

Genetic markers

X-ray responsea

W3110 P3478 AB2500

nic, thy nic, thy, polAl thi, thy, arg, pro, his,

Resistant Sensitive Resistant

Resistant

Sensitive

94 89 91

X9247

leu, uvrA thy, recA, recB

Sensitive

Sensitive

5

responseb

Moderately sensitive

(% survival)

to Town et al. (21). According to Kanner and Hanawalt (10). TP sensitivity was measured by incubation of log-phase cells in TP-M9 medium (equivalency of 150 ,g of unirradiated tryptophan per ml) for 30 min with subsequent dilution and plating on nutrient agar plates, which were incubated 48 h to determine the number of survivors. Greater than 85% survival indicated resistance, and less than 10% survival indicated sensitivity. Since log-phase rec cells are most sensitive to TP in the first cycle of killing and percent survival below 1 to 10% is not exponentially reduced, only the first cycle of killing was used to determine toxicity (22). a According

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fractions were collected from the bottom of the gradients onto Whatman 17 paper strips by the method of Carrier and Setlow (1). The strips were washed once with 5% trichloroacetic acid and twice with 95% ethanol before being dried and counted as above.

RESULTS Effect of TP on DNA replication. The rates of [3Hlthymidine incorporation into trichloroacetic acid-insoluble counts in the presence and absence of TP are shown in Fig. 1. TP had a differential effect on DNA replication for W3110 wild-type, AB2500 uvrA, P3478 polAl, and X9247 recA recB cells. All strains tested slowed or stopped DNA synthesis immediately in the presence of TP. However, the effect of TP on DNA synthesis in W3110 wild-type and AB2500 uvrA cells was only temporary, and the cells recovered a normal rate of DNA replication within 60 min of incubation in the presence of TP. In contrast, TP immediately terminated DNA replication in P3478 polAl and X9247 recA recB, and no recovery of DNA synthesis was observed for these strains during the 150min incubation period. Although the pol and rec mutants are deficient in two separate repair functions, both classes of mutation result in X-ray sensitivity and a reduction in the ability to repair X ray-induced single-strand breaks (21). Since the incubation of log-phase P3478 polAl cells in TP (at a concentration which kills about 90% of a rec population) resulted in less than 10% lethality for polAl mutants (Table 1), the effect of TP incubations on the polAl strain is not lethal, and removal of the photoproduct allowed the cell to divide normally. The constancy of the "C prelabel throughout the TP incubation of all strains tested (Fig. 1) indicates that TP did not initiate degradation of parental DNA and that concurrent DNA degradation did not account for the depression of DNA synthesis in the presence of TP. Moreover, the selective inhibition of DNA synthesis in mutants which are both sensitive to X irradiation (recA, polAl) and reduced in their capability to repair X ray-induced single-strand breaks (21) suggests that TP may affect DNA replication by slowing the repair of replication gaps. Effect of TP on cell division. During the DNA synthesis experiments described above, samples were removed to be counted and sized on the Coulter counter. Except for the X9247 recA recB mutant, cell division and DNA replication remained coupled in the presence of TP (Fig. 2). The response of DNA synthesis (Fig. 1) and rate of increase in cell number to TP (Fig.

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TIME, min FIG. 1. Effect of tryptophan photoproduct (TP) on DNA synthesis of log-phase cells, as determined by the incorporation of [3H]thymidine into trichloroacetic acid-insoluble counts. The response of the following strains of E. coli is shown above: (A) W3110, thy, wild type; (B) AB2500 thy, uvrA; (C) P3478 thy, polAl; (D) X9247 thy, recA, recB. Symbols: A, amount of '4C-prelabeled DNA present as trichloroacetic acid-insoluble radioactivity after the TP-M9 medium incubation; *, amount of [9H]thymidine incorporated into trichloroacetic acid-insoluble radioactivity during the TP-M9 medium incubation; x, amount of [3H]thymidine incorporated into trichloroacetic acid-insoluble radioactivity during a parallel incubation in M9 medium with no TP present.

2) were parallel in all strains tested except the recA recB double mutant. Although DNA synthesis was inhibited in the polAl mutant, there was no increase in cell numbers after 30 min

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(0.16 increase) in the presence of TP. However, during the 150-min TP incubation of the X9247 recA recB mutant, approximately 0.46 cells completed a division with no DNA synthesis to provide complete chromosomes for these cells. Also, the recA recB mutant increased in cell numbers throughout the 150-min TP incubation period (Fig. 2F), whereas DNA synthesis was completely inhibited. Inouye had previously reported that inhibition of DNA replication in E. coli recA cells results in the uncoupling of DNA replication from cell division (9). Our reI

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B. UvrA

sults agree with those of Inouye (9) and indicate that TP inhibition of DNA synthesis in the recA recB mutant resulted in uncoupling of cell division from DNA replication. The cell sizing data in Fig. 3 also indicate that chromosome replication and cell division were uncoupled only in the rec mutant. The wild-type and uvrA cells increased in size during the first 30 min of incubation in TP-M9 medium. However, wild-type and uvrA cells regained a size similar to that of the cells incubated in M9 medium during the TP incuI

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TIME, min FIG. 2. Effect of tryptophan photproduct (TP) on the increase in relative cell numbers of log-phase cells, as measured by total count with a modified Coulter counter. The response of the following strains of E. coli is shown above: (A) W3110 thy, wild type; (B) AB2500 thy, uvrA; (C) P3478 thy, polAl; (D) X9247 thy, recA, recB; (E) W3110 and AB2500 incubated in TP-M9; (F) P3478 and X9247 incubated in TP-M9. Symbols: 0, increase in cell number of W3110 when incubated in TP-M9 medium; A, cell numbers of AB2500 incubated in TP-M9 medium; *, plot of the increase in cell numbers of P3478 in the presence of TP; *, response of increase in cell numbers for X9247 incubated in TP-M9 medium; x, relative increase in cell numbers of parallel samples incubated in M9 medium with no TP present.

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YOAKUM ET AL.

66

A Wildtype W3110

B uvrA AB2500

A,_\ I/N

C

polAl P3478

Iv D RecA RecB X9247

2v

2v IV IV 2V FIG. 3. Effect of tryptophan photoproduct (TP) on the cell size distribution of log-phase cells, as measured with a modified Coulter counter. The response of the following strains of E. coli is shown above: (A) W3110 thy, wild type; (B) AB2500 thy, uvrA; (C) P3478 thy, polA1; (D) X9247 thy, recA, recB. The size distributions of cells incubated for 0, 60, 90, and 120 min in TP-M9 medium are plotted above for each of the strains tested as ;(2) 30 min of incubation in TP-M9 (---;90 min of incubation follows: (I) zero time of incubation ( ( .... ); and 120 min of incubation (- --

bation period. This recovery of normal size distribution parallels the recovery of DNA synthesis in these strains in the presence of TP. The polAl mutant showed no change in cell size during the TP incubation, paralleling the complete inhibition of DNA synthesis and absence of residual cell division observed for the polAl strain. However, the mean cell size of the recA recB mutant increased throughout the TP incubation period even though DNA synthesis was completely inhibited in this strain. Thus, the cell sizing data in Fig. 3 support the conclusion that TP inhibition of DNA synthesis in the recA recB mutant resulted in uncoupling of DNA replication from cell division. Effect of TP on parent and nascent DNA. Figure 4 shows the sedimentation profiles of "C-prelabeled DNA, the DNA labeled with [3H Ithymidine in the presence of TP, and the

chase profiles of the 3H-labeled DNA. Intermediate pieces of 3H-labeled DNA of approximately 7 x 106 daltons (as calculated by using T4 DNA as a marker [5, 13 ]) were observed after labeling in the presence of TP for 15 min (Fig. 4, triangles). These DNA intermediates did not result from TP-activated nucleases since the "C-prelabeled DNA peaked in the same position as control experiments not involving the use of TP (data not shown). Thus, there is no detectable degradation of parental DNA in wild-type cells during the TP incorporation and chase incubations. The appearance of small pieces of DNA corresponding in size to late replicative intermediates reported by Okazaki and others in E. coli (18), in cells allowed to incorporate [3H ]thymidine for 15 min in the presence of TP, could be explained by the following hypotheses.

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(A) TP could cause the accumulation of replicative intermediates that would normally be rapidly joined into high-molecular-weight DNA by inhibiting a step(s) required for the closure of these gaps; (B) TP might interfere directly with the replication fork by intercalating between strands in front of the replication fork, producing a gap in the nascent DNA; (C) TP might act by stimulating a nuclease which degrades the DNA into pieces of approximately 7 x 108 daltons. To distinguish among these possibilities, chase times of nascent DNA pieces synthesized in the presence of TP into high-molecularweight DNA were compared for samples chased in the presence and absence of TP. The nascent DNA pieces which were chased in TP-M9 medium required 5 min of incubation before the DNA pieces were returned to the same position on the alkaline sucrose gradient as the TCprelabeled parental DNA (Fig. 4, diamonds). In contrast, samples chased without TP present required only 60 s of chase time to regain the control position on the alkaline sucrose gradient

I

600

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'3

400

0

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67

(Fig. 4, squares). A comparison of these chase times indicates that TP causes an 80% reduction in the rate of gap-closing events. This result is consistent with the first hypothesis, that the appearance of low-molecular-weight DNA in cells labeled in the presence of TP represents the accumulation of replicative intermediates as a result of the inhibition of some step(s) in the closing of these gaps. DISCUSSION It is clear from the following facts that TP has a differential effect on DNA replication in mutants sensitive to single-strand gaps in DNA: (i) DNA replication in wild-type W3110 cells first slowed and then resumed a normal rate during the TP incubation; (ii) uvrA AB2500 cells slowed DNA replication and recovered in a similar manner; (iii) the polAl mutant P3478 stopped DNA replication immediately in the presence of TP; and (iv) the recA recB double mutant stopped DNA replication immediately I

I

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50 0

Photoproduct Pulse- Medium Chase Photoproduct Pulse- Photoproduct Chase .'14C Prelabel 14C Prelabel * 15 min TP-labeling W31 10 w ildtype A 15 min TP-labeling * 2 min Chase o 30 sec Chase 400 * 3 min Chase 13 60 sec Chase O 5 min Chase 300 -

a.0 f)300-

a.

FRACTION NUMBER sucrose sedimentation Alkaline FIG. 4. gradient profiles of DNA from W3110 thy wild type, labeled or treated as follows: (i) DNA that was prelabeled with [14C]thymine in M9 medium, from cells which were then washed and incubated in TP-M9 medium with [3Hlthymidine for 15 min; (it) DNA that was synthesized in TPM9 medium labeled with [3H]thymidine; (iii) DNA that had been synthesized inTP-M9 mediumand then chased in either M9 medium or TP-M9 medium with cold thymidine.

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YOAKUM ET AL.

in the presence of TP. Therefore, DNA replication is especially sensitive to TP in mutants (polAl, recA recB) deficient in the repair of single-strand breaks in their DNA. The special sensitivity to TP of DNA replication in these mutants suggests the possibility that TP inhibition of a repair function required for rapid gap closure, other than DNA polymerase I activity, could be responsible for the observed effect on DNA replication. There are several steps at which inhibition of a gap repair function might selectively stop DNA synthesis in mutants sensitive to single-strand breaks. These steps include: the inhibition of a DNA polymerase other than the polAl gene product (i.e., pol II or pol III); the inhibition of an RNA nuclease disallowing removal of the primer; or the inhibition of DNA-ligase-mediated joining of nascent pieces. Inhibition of replication gap closure at one or more of these steps would be expected to inhibit DNA replication more effectively in mutants defective in recA or polAl activity than in cells with normal gap repair abilities. Inhibition of DNA replication in E. coli B/r normally allows only a 25% residual increase in cell numbers (2, 7) or causes immediate termination of cell division (4, 8), as we observed for the polAl mutant. However, Inouye has reported that inhibition of DNA replication (by nalidixic acid or thymine starvation) in an E. coli recA strain results in more than 25% residual division, an increase in mean cell size, and uncoupling of DNA replication from cell division (9). Inouye attributes this uncoupling to a pleiotrophic effect of the recA mutation on cell division (9). The effect of TP-mediated inhibition of DNA synthesis on cell division in the recA recB and polAl mutants is in agreement with this finding. The conclusion that TP inhibition of DNA synthesis in the recA recB mutant results in uncoupling is supported by the following facts: (i) TP inhibition of DNA replication in the polAl mutant caused cell division to terminate within 30 min but did not cause an increase in mean cell size; (ii) in contrast, TP inhibition of DNA replication in the recA recB mutant did not terminate cell division and there was a steady increase in cell size throughout the TP incubation; (iii) the viability of the rec mutant was substantially reduced during the treatment, whereas the pol strain remained viable during the TP incubation. The analysis of prelabeled DNA and nascent DNA synthesized in TP-M9 medium on alkaline sucrose gradients provides evidence that TP inhibits some step(s) necessary for closing replication gaps. Since even late replication intermediates are difficult to detect in wild-type

J. BACTERIOL.

cells growing in glucose at 37 C (18), the appearance of nascent pieces of DNA during the first 15-min TP labeling period suggests that TP might inhibit replication gap closure. This lowmolecular-weight nascent DNA also could result from direct TP interference with the replication fork if TP acts on the DNA in front of the replication fork [hypothesis (B) ] or by TP stimulation of nuclease [hypothesis (C) ]. If TP caused extra gaps in nascent DNA via intercalation between strands of DNA in front of the replication fork, then the chase time of these gaps should be the same whether in the presence or absence of TP. This explanation is not consistent with the observation that TP slows the chase of small pieces into control-size DNA by 80%. If the pieces of DNA which appear during TP incubation were the result of TPstimulated nucleolytic activity (hypothesis C), then the '4C-prelabeled DNA would not sediment to the control DNA position in the alkaline sucrose gradient. If TP stimulated the action of a "daughter-specific" nuclease (hypothesis C), then nascent DNA pieces would not be expected to return to high-molecular-weight DNA in the presence of TP. Hypothesis C is not consistent with the observation that "C-prelabeled DNA is not degraded and that pieces of DNA chase into high-molecular-weight DNA in the presence of TP. The observed results are most consistent with hypothesis A: accumulation of nascent DNA pieces during the TP labeling period is caused by TP inhibition of some step(s) of normal replication gap closing. Both recA and polA1 mutations have been shown to result in a special sensitivity to single-strand breaks in their DNA (21), and the polA 1 mutant crossed with a recA or recB mutant produces inviable progeny (6, 15). It has also been shown that polAl cells close replication gaps at 10% of the rate of their pol+ parent (12, 17), even though replication proceeds at the same rate in both strains (3). However, a temperature sensitive po1Al, recA double mutant unexpectedly closes replication gaps at the same rate as the polAltA single mutant when shifted to the nonpermissive temperature (16). Therefore, recA does not appear to be directly involved in gap closing (16). We interpret these observations to suggest that the lethality of the polA 1- condition for rec- cells may be the result of the special sensitivity of the rec mutation to gaps in its DNA. The introduction of the polAl gene into a rec- recipient would be expected to increase the number of replication gaps 10-fold without any reduction in the rate of DNA synthesis (3, 12. 17), and since the rec mutation causes a special sensitivity to gaps in DNA, lethality results. The observation that recA

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gene product cannot directly contribute to the closure of replication gaps (16) suggests that the role of recA may be to maintain conditions which will allow the cell to remain viable in the absence of normal gap-closing rates. Our interpretation that slower rates of replication gap repair are lethal for recA mutants correlates well with the effects we have reported here for TP. This photoproduct affects DNA replication by slowing the repair of replication gaps. Also, the photoproduct is selectively lethal for recombination-deficient mutants and uncouples DNA replication from cell division in recA recB mutants. These observations suggest that the recA gene product plays a regulatory role in replication gap closing. Therefore, we propose the following as a possible explanation of these results: the recA+ gene product senses the number of postreplication gaps by monitoring the configuration of the chromosome. When the number of postreplication gaps increases, the recA+ gene product alters the replication complex to conform to the change in chromosome configuration. In this way, DNA replication would be coordinated with repair of postreplication gaps, and when a stable chromosome configuration is reached (i.e., the rate of appearance of postreplication gaps is constant), DNA replication would resume a normal rate in rec+ cells. However, in the absence of functional recA+ gene product, DNA replication would fail to adjust to any increase in the number of postreplication gaps, and continuation synthesis would stop with the chromosome and replication complex in an unrecoverable configuration. We conclude that the biological specificity of TP for mutants deficient in recombination repair is a result of the special sensitivity of these mutants to gaps in their DNA. Therefore, TP kills log-phase rec- mutants because the photoproduct slows the rate of replication gap repair, increasing the number of postreplication gaps, which is a lethal condition for the cell. ACKNOWLEDGMENTS We thank Tatsuo Matsushita for his critical conversations throughout this work and Kevin McEntee of the Department of Biophysics, University of Chicago, for the recA recB mutant X9247. This work was supported by the Atomic Energy Commission; by a CEA Lab-Grad Participantship; and by National Science Foundation grant GB-33869 to A. E.

LITERATURE CITED 1. Carrier, W. L., and R. B. Setlow. 1971. A paper strip method for assaying gradient fractions containing radioactive molecules. Anal. Biochem. 43:427-432. 2. Clark, D. J. 1968. Regulation of deoxyribonucleic acid replication and cell division in Escherichia coli B/r. J.

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