Mar 12, 1973 - recB Derivatives of an Escherichia coli K-12. Strain with a ... polA12 recB21,continue to incorporate a sub- ... tion, hypothetical "swivel" breaks (21), or gaps between replicative ... 2.4 cm) which were washed once with cold 5%.
JouRNAL OF BACTROLOGY, June 1973, p. 1014-1017 Copyright 0 1973 American Society for Microbiology
Vol. 114, No. 3
Printed in U.SA.
Deoxyribonucleic Acid Synthesis in recA and recB Derivatives of an Escherichia coli K-12 Strain with a Temperature-Sensitive Deoxyribonucleic Acid Polymerase I M. MONK, J. KINROSS, AND C. TOWN
Molecular Genetics Unit, Medical Research Council, Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland Received for publication 12 March 1973
recA and recB derivatives of a strain of Escherichia coli with a temperaturesensitive deoxyribonucleic acid (DNA) polymerase I (polA12) are inviable at high temperature, but continue to incorporate 3H-thymine into DNA for extended periods. The DNA made in pulse-chase experiments at high temperature in the poIA12 parent and its double-mutant derivatives has been examined by alkaline sucrose gradient sedimentation analysis. The low-molecular-weight DNA fragments made during short pulses were joined at the same rate in each strain. Furthermore, the resulting high-molecular-weight DNA was of the same size in each case and was stable for at least 50 min. It is concluded that the inviability of the double mutants is due neither to a defect in converting low-molecular-weight DNA intermediates to high molecular weight nor to the presence of unrepaired random breaks in their DNA.
Gross et al. (6) were not able to construct a double mutant of Escherichia coli that was defective in the rec recombination pathway (recA) as well as in deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7., polA). They concluded that polA recA double mutants are inviable. This was confirmed by Monk and Kinross (13), who found that recA as well as recB derivatives of a strain with a temperaturesensitive DNA polymerase I (polA12) are inviable at 42 C. The nature of the requirement for either DNA polymerase I or the rec gene products for cell growth is not known. It is unlikely that progression of the replication fork (i.e., DNA synthesis per se) is involved, since the conditional lethal strains, polA12 recA56 and polA12 recB21, continue to incorporate a substantial amount of 3H-thymine into DNA at 42 C (13). pol and rec functions are important for the repair of X-ray-induced single-strand breaks in DNA (9, 20). Their role in repair of ultravioletinduced damage can be attributed to "gap repair": there is genetic evidence that DNA polymerase I fills in the gap after dimer excision (14) and that the recA gene product is involved in the repair of single-strand gaps produced by
the replication of DNA containing unexcised dimers (16, 17). It would thus seem feasible that the function necessary for cell survival performed by the polA or rec gene products could be the repair of breaks or gaps in the DNA. Such discontinuities could arise fortuitously during growth or could be specifically associated with DNA metabolism; for example, initiation and termination of rounds of chromosome replication, hypothetical "swivel" breaks (21), or gaps between replicative intermediates (Okazaki fragments; 18). In this paper we examine the rate at which replicative intermediates in the double mutants, pulse labeled at 42 C, move into highmolecular-weight DNA, and the stability of this DNA. The results indicate that the defect in polA12 recA56 and poIA12 recB21 at 42 C is not an accumulation of breaks in DNA. MATERIALS AND METHODS Bacterial strains. The bacterial strains used, isogenic except for the pol, rec, and thy mutations, are listed in Table 1. They are derived from W3110 rhalac- str-r. polAl is an amber mutation and W3110 does not carry an amber suppressor. Media. Cultures were grown in M9 (glucose-salts)
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medium prepared as described by Anderson (1). The medium (MIN-CAS) contained 10-3 M MgSO4 and was supplemented with 0.5% Casamino Acids (Difco). Labeling experiments. Thymidine-methyl-3H (TRK120) and thymine-2-14C (CFA 182) were obtained from Radiochemical Centre, Amersham, England.
Fully 14C-thymine-labeled cells
were
prepared by
at least five generations of growth at 30 C in MINCAS medium supplemented with 100 Ag of deox-
yguanosine per ml and 1 Mg of "4C-thymine per ml (sp act 56 mCi/mM). For the thy- strain, "4C-thymine was added to 5 ,g per ml. Pulse labeling. Log-phase cells growing in MINCAS medium supplemented with 100 Ag of deoxyguanosine per ml (and with 5 MAg of thymine per ml for the thy- strain) at 30 C were transferred to 42 C for 15 min and then pulse labeled by the addition of 3H-thymidine to a concentration of 1 Mlg per ml (sp act 26 Ci/mM). The pulse was terminated after 60 s by pouring the sample into approximately four volumes of "stop mix" (0.01 M KCN, 0.01 M ethylenediaminetetraacetic acid (EDTA), 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.1, and 1,000 of thymidine per ml) at 0 C.
Mg
Pulse chase. The pulse was terminated by the addition of nonradioactive thymidine to a concentration of 1,000 Mg/ml (1,000-fold excess). The pulse was chased by further incubation at 42 C for times as indicated in the figure legends, and a 0.1-ml sample was transferred to 0.4 ml of stop mix at 0 C. The time in stop mix (between 15 and 50 min) did not affect the subsequent sedimentation pattern of the DNA. The stop mix was shown to stop further incorporation of 3H-thymidine into DNA in less than 10 s, the earliest time a sample could be removed. Alkaline sucrose gradients. Linear alkaline sucrose gradients (12) from 5 to 20% sucrose (wt/vol) in 0.3 N NaOH and 0.01 M EDTA were made by using a Buchler gradient maker with a peristaltic pump. Samples (0.05 ml) of "4C-thymine- and 3H-thymidinelabeled cells from the stop mixes (5 x 10" total cells) were layered on top of the 4-ml gradient in a 0.2-ml cap of 0.5% Sarkosyl (Geigy NL30) in 0.5 N NaOH and 0.01 M EDTA. The cap was gently stirred with a pin. The gradients stood at room temperature for 15 min before centrifugation at 30,000 rpm at 20 C for 105 min in an SW39 rotor in a Spinco model L ultracentrifuge. After centrifugation, eight-drop fractions were collected on Whatman filter papers (3 MM, 2.4 cm) which were washed once with cold 5% trichloroacetic acid and twice with alcohol, and air dried. The filters were transferred to 5 ml of scintillation fluid [0.3% 2, 5-diphenyloxazole, 0.03% 1, 4-bis-2(5-phenyloxazolyl)-benzene in toluene] and counted in a Packard scintillation counter. Phage A lightly labeled with "4C-thymine, kindly supplied by R. Bird, replaced the "4C-thymine-labeled E. coli DNA in one gradient in each run. RESULTS
DNA polymerase I is temperature sensitive in strains carrying the mutation polA12. We there-
fore followed the rate of conversion of 3H-thymidine-labeled replicative intermediates into higher-molecular-weight DNA at 42 C in the strains listed in Table 1. DNA fully labeled with "4C-thymine in a parallel culture grown at 30 C was used as standard high-molecular-weight DNA. The experimental details and results are given in Fig. 1. The pol rec double mutants incorporated fewer counts in 60 s, in keeping with their slow growth rate (13). The high proportion of counts appearing at the top of the gradients for these strains are not relevant, since a comparable number of counts are seen in the last few fractions for gradients of DNA labeled with short pulses in all strains. A comparison of Fig. la, lb, and lc shows that DNA labeled for 60 s at 42 C is converted into high-molecular-weight DNA in the pol rec double mutants in a manner similar to that observed in the polA12 single mutant. After a 20-min chase at 42 C, the pulse-labeled single-strand DNA sediments close to the position of fully "4C-labeled singlestranded DNA made at 30 C. Fig. ld shows, for polA12 recA56, that the pulse-labeled DNA does become completely heavy at 42 C and remains stable for at least 50 min at 42 C, the longest chase examined. Similar results (unpublished data) were obtained for polA12 and polA12 recB21. The molecular weight of the DNA synthesised in a 60-s pulse is much greater in a pol+ cell (Fig. lf) than in any of the polA mutants. However, the molecular weight of the smaller pieces (Okazaki fragments) made in a 10-s pulse is the same in both pol+ and polA strains (Monk and Kinross, data not shown; see also reference 11). The joining of these pieces is thus slower in poIA12 and polAl strains than in a pol+ strain, regardless of their rec genotype.
DISCUSSION pol and rec functions are important in the handling of breaks or gaps artificially induced in DNA chains by treatment of cells with ultraviolet rays or X-rays (8, 9, 17, 20). One TABLE 1. Escherichia coli strains No.
JG138 MM385 MM386 MM387 MM451
Charactersa thy-polAl thy+ po1A12recA56 thy+polA12 thy+polA12recB21 thy+
Reference
14 13 13 13 M. Monk
a Symbols for genetic markers are as listed by Taylor (19).
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MONK, KINROSS, AND TOWN
Fraction
J.BACTERIOL.
Number
FIG. 1. Alkaline sucrose gradient sedimentation of pulse-labeled DNA. For each strain, parallel cultures, one fully labeled with "4C-thymine and one unlabeled, were set up at 30 C and the optical density followed. Experiments were initiated approximately 40 min (a, b, c, e, f) or 60 min (d) before the optical density reading reached a value corresponding to approximately 2 x 108 cells per ml. The unlabeled culture was shifted to 42 C and 15 min later was pulse labeled for 60 s with 3H-thymidine. In a, b, c, e, and f, the pulse was terminated by ) or by the addition of 1,000-fold excess of cold thymidine and pouring a sample into stop mix (chase 0 min, continued incubation at 42 C followed by sampling into stop mix (chase 5 min, . chase 20 min ......... Due to the different methods of terminating the pulse, the total counts for the unchased and chased samples are not comparable. In d the chase times were 20 min ( ), 35 min (-----), and 50 min ( ........ ). Fully "C-labeled DNA from cells grown at 30 C is shown by the heavy line ( ). The arrow depicts the sedimentation of X DNA. Sedimentation is from right to left. The experiments reported in a and f were repeated using two volumes of acetone at -20 C as stop mix, with essentially the same results. Further details of labeling and sedimentation in alkaline sucrose gradients are given in Materials and Methods.
plausible explanation for the conditional lethality of polA12 recA56 and polA12 recB21 is that breaks or gaps in the DNA accumulate at 42 C when both the rec function concerned and DNA polymerase I are absent. Our results (Fig. 1) show that DNA made at 42 C in the double mutants is ultimately converted to highmolecular-weight DNA and remains stable even 65 min after the shift to 42 C in polA12 recA56 (Fig. ld) where extensive DNA degradation
occurs at the high temperature (13). In this connection, it is interesting that DNA breakdown does not generate a diversity in size of single-strand DNA. This suggests that the degradation must be an all or nothing phenomenon, essentially removing the DNA strand concemed. We have therefore found no evidence for an accumulation of breaks in the double mutants at 42 C. However, calculations based on the Burgi-Hershey relationship (2) indicate that
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DNA SYNTHESIS IN pol-rec- E. COLI
we cannot exclude the possibility of up to three breaks per chromosome. Both Kuempel and Veomett (11) and Okazaki et al. (15) observed a slow joining of replicative intermediates in the original polAl strain (3, 5) compared with pol+ strains! We confirmed their observations for polAl and extended them to poIA12 at 42 C. We considered the possibility that poIA12 rec double mutants are inviable at 42 C due to an exaggeration of this defect, but the experiments reported (Fig. 1) clearly demonstrate the same rate of conversion of pulse-labeled replicative intermediates into high-molecular-weight DNA in the double mutants as in the single polA12 mutant. The problem of the lethality of pol rec double mutants remains unsolved. To date, the only defect we observed is dependence on cell density for growth at 30 C and aberrant morphology at 42 C, namely, filamentation, localized swellings, and vacuolation. We previously reported (13) that extensive DNA breakdown occurs at 42 C in polA12 recA56 but not in polA12 recB21. We constructed the triple mutant poIA12 recA56 recB21. It is also inviable at 42 C and does not break down its DNA (Monk and Kinross, unpublished data). There is accumulating evidence implicating the recA function in some cellular control mechanism coordinating cell division with DNA metabolism (4, 7, 10). The possibility remains that the essential requirement for pot or rec function for growth exists at the level of such control systems, such as the timing of initiation of DNA replication or segregation of completed chromosomes.
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LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virusresistant mutants of Escherichia coli strain "B." Proc. Nat. Acad. Sci. U.S.A. 32:120-128. 2. Burgi, E., and A. D. Hershey. 1963. Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3:309-321. 3. de Lucia, P., and J. Cairns. 1969. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1164-1166. 4. Green, M. H. L., J. Greenberg, and J. Donch. 1969. Effect of a recA gene on cell division and capsular polysaccha-
18.
19. 20. 21.
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ride production in a ton strain of Escherichia coli. Genet. Res. 14:159-162. Gross, J. D., and M. Gross. 1969. Genetic analysis of an Escherichia coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1166-1168. Gross, J. D., J. Grunstein, and E. M. Witkin. 1971. Inviability of recA- derivatives of the DNA polymerase mutant of de Lucia and Cairns. J. Mol. Biol. 58:631634. Inouye, M. 1971. Pleiotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid replication. J. Bacteriol. 106:539-542. Kanner, L., and P. Hanawalt. 1970. Repair deficiency in a bacterial mutant defective in DNA polymerase. Biochem. Biophys. Res. Comm. 39:149-155. Kapp, D. S., and K. C. Smith. 1970. Repair of radiationinduced damage in Escherichia coli. [l. Effect of rec and uvr mutations on radiosensitivity, and repair of X-ray-induced single-strand breaks in deoxyribonucleic acid. J. Bacteriol. 103:49-54. Kirby, E. P., F. Jacob, and D. A. Goldthwaite. 1967. Prophage induction and filament formation in a mutant strain of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 58:1903-1910. Kuempel, P. L., and G. E. Veomett. 1970. A possible function of DNA polymerase in chromosome replication. Biochem. Biophys. Res. Comm. 41:973-980. McGrath, R. A., and R. W. Williams. 1966. Reconstruction in vivo of irradiated Escherichia coli deoxyribonucleic acid; the rejoining of broken pieces. Nature (London) 212:534-535. Monk, M., and J. Kinross. 1972. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J. Bacteriol. 109:971-978. Monk, M., M. Peacey, and J. D. Gross. 1971. Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells. J. Mol. Biol. 58:623-630. Okazaki, R., M. Arisawa, and A. Sugino. 1971. Slow joining of newly replicated DNA chains in DNA polymerase I-deficient Escherichia coli mutants. Proc. Nat. Acad. Sci. U.S.A. 68:2954-2957. Rupp, W. D., and P. Howard-Flanders. 1968. Discontinuities in the DNA synthesised in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31:291-304. Smith, K. C., and D. H. C. Meun. 1970. Repair of radiation-induced damage in Escherichia coli 1. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51:459-472. Sugino, A., S. Hirose, and R. Okazaki. 1972. RNA-linked nascent DNA fragments in Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 69:1863-1867. Taylor, A. L. 1970. Current linkage map of Escherichia coli. Bacteriol. Rev. 34:155-175. Town, C. D., K. C. Smith, and H. S. Kaplan. 1971. DNA polymerase required for rapid repair of X-ray-induced DNA strand breaks in vivo. Science 172:851-854. Wemer, R. 1971. Mechanism of DNA replication. Nature (London) 230:570-572.
