JOURNAL OF BACTERIOLOGY, May 1979, p. 486-491 0021-9193/79/05-0486/06$02.00/0
Vol. 138, No. 2
Ionizing Radiation Damage to the Folded Chromosome of Escherichia coli K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid KEVIN M. ULMER,t* REINALDO F. GOMEZ,' AND ANTHONY J. SINSKEY' Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139' and Joint Program in Biological Oceanography, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 7 September 1978
'The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475-485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of doublestrand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.
Until recently, the available evidence had supported the belief that ionizing radiation-induced DNA double-strand breaks in Escherichia coli are lethal lesions not subject to repair (1, 8). Direct evidence of repair of double-strand breaks has been reported for more radiationresistant bacteria, however. Significant amounts of repair occur in Micrococcus radiodurans (3, 4, 9, 10) and in a highly radiation-resistant strain of Salmonella typhimurium LT2 developed by Davies and Sinskey (5, 6; D. Baraldi and A. J. Sinskey, unpublished data). The detailed mechanisms of repair in these strains have not been elucidated, however, largely due to the lack of suitable, well-characterized repair-deficient mutants. These earlier studies were also plagued by rotor speed effects (14) and by problems associt Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139.
ated with cell lysis and DNA extraction. Shearing during the lysis procedure was unavoidable, and the largest pieces of unirradiated DNA that could be obtained routinely represented only a fraction of the molecular weight of the intact genome (2). Recent technical improvements in these areas have enabled two groups to demonstrate doublestrand break repair in E. coli. Krisch et al. (13) produced DNA double-strand breaks by incorporating 125I into the DNA and allowing for decay. Incubation of damaged cells in growth medium resulted in repair of up to three or four double-strand breaks per genome in the wildtype (WT) strain (strain AB2497), whereas no repair was observed in a recA13 strain (strain AB2487). There was a one-to-one correlation between cell killing and the number of postincubation residual double-strand breaks. With the same E. coli strains, Krasin and Hutchinson
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(12) have obtained quite similar results by using gamma irradiation in an oxygenated solution to produce the double4strand breaks. By using very low gamma ray doses, they have demonstrated that double-strand break repair requires the presence of multiple genome equivalents of DNA per cell as well as functional recA and recB genes. In the present study, using procedures for the gentle isolation and unfolding of nucleoid DNA (17), we demonstrate directly rec-dependent repair of several double-breaks per genome and present evidence for recombinational intermediates involved in this repair process. MATERIALS AND METHODS The procedures for cell growth, labeling, irradiation, nucleoid isolation and unfolding, and sedimentation analysis have been presented in detail in another paper (17). Survival after gamma irradiation. E. coli strains AB2497 WT and AB2487 recA13 were grown overnight in M9 minimal medium. Fresh 20-ml cultures were inoculated with 0.2 ml of the overnight cultures, grown with shaking at 37°C for 4.5 to 5 h, and then quickly cooled in ice water and harvested by centrifugation at 7,500 rpm for 5 min. The cells were washed with 20 ml of M9 salts plus 10,ug of thymine per ml, centrifuged a second time, and finally resuspended in 12 ml of M9 salts plus thymine. Samples were held on ice in glass test tubes (12 by 75 mm) during irradiation. Dilutions were made in phosphate buffer, and the cells were plated in duplicate (0.1 ml/ plate) on M9 minimal medium plates (15 g of agar [Difco] per liter). These were incubated at 37°C for 48 h before colonies were counted.
RESULTS Survival curves for gamma-irradiated E. coli K-12 strains. Gamma irradiation of exponential-phase cells of strains AB2497 WT and AB2487 recA13 at 00C in M9 buffer under aerobic conditions resulted in the survival patterns indicated in Fig. 1. The points shown are the averages of the results of three separate experiments and indicate that for the WT strain, there is an exponential decrease in survival for doses up to 100 krad. The markedly enhanced sensitivity of the recA13 strain irradiated under identical conditions is also apparent. Repair of gamma radiation damage: folded chromosomes. When strain AB2497
WT was incubated at 370C in complete M9 medium after gamma irradiation in M9 buffer at 0°C, major changes occurred in the gradient profiles of folded chromosomes. Figure 2 demonstrates the effects of post-irradiation incubation after a dose of 10 krad. Immediately after irradiation, the nucleoids were shifted toward the top of the gradient, and the sedimentation
487
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FIG. 1. Survival of E. coli strains AB2497 WT on minimal medium plates after gamma irradiation in M9 buffer.
(0) and AB2487 recA13 (0)
CL
FRACTIONAL DISTANCE SEDIMENTED
FIG. 2. Gradient profiles for repair of gamma-irradiated (10 krads) folded chromosomes of E. coli strain AB2497 WT. Gradients were centrifuged at 2,949 rpm, for a total integrated centrifugal force (42t) of 6.00 x 109 rad'/s. (A) Symbols: 0, pre-irradiation; 0, immediately post-irradiation; A, 0.5-h incubation. (B) Symbols: 0, 1-h incubation; 0, 2-h incubation; A, 3-h incubation.
coefficient was reduced from 1,762S to only 1,370S. After 0.5 h in growth medium, the entire peak shifted to 2,050S, which is in the range of the most rapidly sedimenting nucleoids in the original, unirradiated profile. The sedimentation rate continued to increase, reaching 2,318S after 1 h. The material was more heterogeneous than that of the unirradiated profile, and the peak
ULMER, GOMEZ, AND SINSKEY
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was broadened accordingly. After 2 h of incubation, the peak sharpened more, and the material moved back toward the top of the gradient to a position at the low-S side of the unirradiated distribution (1,686S). Finally, after 3 h, the nucleoids once again shifted slightly toward more rapidly sedimenting material, returning to the sedimentation rate of unirradiated nucleoids (1,793S). With increasing length of incubation, increasing amounts of rapidly sedimenting material appeared in the last few fractions at the bottom of the gradients as well. Any material in the last fraction at the bottom of the gradient had a sedimentation coefficient greater than 3,100S, which is the range observed for membrane-associated nucleoids (15, 16). When strain AB2487 recA13 was incubated at 370C in complete M9 medium after receiving a dose of 5 krad in M9 buffer at 00C, the gradient profiles for nucleoids were significantly different from those observed with the WT strain (Fig. 3). Immediately after irradiation, the peak shifted toward the top of the gradient, as was observed previously, but upon incubation the sedimentation coefficients of the material continued to decrease until most of the material was at the very top of the gradient. No rapidly sedimenting material was formed during incubation, as revealed by the total absence of radioactivity below the position of the unirradiated peak in the 10 to 50% gradients used for this experiment. A high-density shelf was included in these gradients as well, to detect any very rapidly sedimenting material that might be lost to the bottom of the tube. The only indication of possible repair was the presence of a small shoulder on
J. BACTERIOL.
the 3-h incubation profile at the right-hand side of the initial, unirradiated peak. Repair of gamma radiation damage: RNase-unfolded chromosomes. When nucleoids from AB2497 WT were unfolded by digestion with RNase after post-irradiation incubation in M9 complete medium, gradient profiles like those in Fig. 4 were obtained. A large decrease in the sedimentation rate was observed immediately after irradiation. The sedimentation coefficient for the unirradiated nucleoids was 1,098S, which was reduced to only 786S after a 5-krad irradiation. Return ofthis material to the position of unirradiated material in the gradients was quite rapid, as indicated by the 0.5-h incubation profile (1,042S). This material was fairly heterogeneous, and included a range of sedimentation coefficients on either side of the unirradiated nucleoid peak. With further incubation, the profile sharpened, and by 3 h the width was the same as that of the profile of unirradiated chromosomes. The mean sedimentation coefficient after 3 h of incubation (1,133S) was slightly increased over the value for unirradiated DNA. The extent of repair after 2 h of post-irradiation incubation at several doses of gamma rays
0
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25
2 2
0.
E 5,
A
0.4 0.2 0.6 0.8 FRACTIONAL DISTANCE SEDIMENTED
FIG. 3. Gradient profiles for repair of gamma-irradiated (5 krads) folded chromosomes from E. coli strain AB2487 recA13. Gradients were centrifuged at an average rotor speed of 2,781 rpm, for a total integrated centrifugal force (w2t) of 6.00 x l0o rad2/s. Symbols: v- *, pre-irradiation; 0-0, immediately post-irradiation; A A, 0.5-h incubation; v X, 3-h incubation. -
0.8 0.4 0.6 0.2 FRACTIONAL DISTANCE SEDIMENTED
FIG. 4. Gradient profiles for repair of RNase-unfolded chromosomes of E. coli strain AB2497 WT after a 5-krad gamma irradiation. The gradients were centrifuged at an average rotor speed of about 3,000 rpm, for a total integrated centrifugal force (W2t) of 9.00 x 109 rad2/s. (A) Symbols: 0, unirradiated; 0, immediately post-irradiation; A, 0.5-h incubation. (B) Symbols: *, 1-h incubation; 0, 2-h incubation; A, 3-h incubation.
ESCHERICHIA CHROMOSOME REPAIR
'VOL. 138, 1979 determined in a final experiment (Fig. 5). Complete recovery occurred after doses of 0.5 to 2.0 krads within this 2-h incubation period, whereas some material with lower sedimentation coefficients was still present in the profiles for 5.0 and 10.0 krads. This is consistent with the results shown in Fig. 4, where 3 h of incubation was required before all of the low-S material had been repaired. That fraction of the material that did not return to the position for unirradiated chromosomes appeared to sediment to the very bottom of the gradient, although some small amount of material also appeared at the top of the 5- and 10-krad profiles. The percentage of material that did return to the position of unirradiated chromosomes is indicated in Table 1, along with the percent survival at the same doses from Fig. 1. The agreement between the two sets of numbers is quite good, suggesting that the percentage of RNase-unfolded chromosomes repaired was equivalent to the percentage of cells that survived.
was
DISCUSSION Repair of gamma radiation damage to folded chromosomes. When E. coli WT cells were incubated for 2 to 3 h in complete growth medium at 370C after gamma irradiation in buffer at 0°C under aerobic conditions, significant amounts of material returned to the posiIC0
A
2 6
4
2
a. B
FRACTIONAL DISTANCE SEDIMENTED
FIG. 5. Gradient profiles for 2-h repair of RNaseunfolded chromosomes of E. coli strain AB2497 WT after several doses of gamma rays. Centrifugation was carried out at an average rotor speed of 3,584 rpm, for a total integrated centrifugal force (w2t) of 9.00 x 109 rad2/s. (A) Symbols: 0, unirradiated; 0, 0.5 krad; A, 1.0 krad. (B) Symbols: 0, 2.0 krads; 0, 5.0 krads; A, 10.0 krads.
489
TABLE 1. Recovery of RNase-digested nucleoids after 2 h of post-irradiation incubation, and cell survival Dose (krad)
% nucleoids' Recovery of
% Survivalb
0.5 1.0 2.0 5.0 10.0
98 93 81 79 41
95 91 83 63 40
a From Fig. 5. The major peaks obtained after 2 h of repair were integrated and expressed as a percentage of the integrated peak for unirradiated chromozomes. b From Fig. 1.
tion of unirradiated nucleoids in the gradients. This behavior was not observed after irradiation of a recA13 strain, suggesting that repair of folded chromosomes may require recombination. The 2,300S peak observed after the fit hour of incubation may represent a recombinational intermediate, since it is in the range of sedimentation coefficients of nearly replicated, double chromosomes (11). The kinetics of its appearance rule out the possibility that direct, radiation-induced cross-linking is responsible for its formation. Subsequent shifts in the profile may indicate segregation of daughter chromosomes before the onset of normal replication and division. Several researchers have reported the formation of rapidly sedimenting, membrane-associated material during repair of ionizing radiation damage (3, 4; Baraldi and Sinskey, unpublished data). In the present experiments, during the later stages of post-irradiation incubation, large amounts of material with sedimentation coefficients in excess of 3,100S were formed. This is within the range of sedimentation rates observed for membrane-associated nucleoids (16) and suggests that radiation-induced cell death may result from the inability of cells to properly segregate daughter chromosomes from the membrane (7). Repair of double-strand breaks. The problem that has confronted all previous investigations of repair of double-strand breaks in E. coli has been to measure viability and the rate of formation of double-strand breaks in the same biologically significant dose range. Unfortunately, this has generally not been possible. The background level of double-strand breaks accumulated during cell lysis precluded measurement of repair of less than six or seven doublestrand breaks per genome (1, 8). In an attempt to circumvent this shortcoming, the number of breaks expected in the biologically significant dose range was estimated by extrapolation from
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measurements of breakage at higher doses and compared with the number of lethal hits determined from survival curves at the lower doses. The most recent calculations of this type indicate that 1.3 to 1.4 double-strand breaks occur per lethal event (2). Our procedure for isolating nucleoids yielded minimally damaged DNA. Few, if any, singlestrand breaks occurred during isolation, as evidenced by the presence of supercoiling, and when nucleoids were examined under an electron microscope, no free ends were observed, indicating that no double-strand breaks were formed. The gentle unfolding techniques that we have developed (RNase or heat) yielded DNA of very high molecular weight, essentially eliminating the problem of background breaks and permitting direct measurement of doublestrand break formation by gamma rays in the biologically significant dose range of less than 10 krads. An average of one lethal event per cell corresponds to a survival of 37%, which occurs at a dose of 10 krads for strain AB2497 (Fig. 1). The average number of double-strand breaks per genome at this dose is 2.7, based on data from our experiments (17), or 3.7, based on the data of Lyderson and Pettijohn (15) for in vivo gamma irradiation of nucleoids. The absolute values of these figures are still subject to question, however, due to uncertainties surrounding the initial conformation of the DNA, and these conclusions can only be considered suggestive of double-strand break repair. The more conclusive evidence of doublestrand break repair comes from experiments reported here, in which post-irradiation incubation of the WT cells resulted in the return of initially broken, RNase-unfolded chromosomal DNA to the same position in the gradient as that of unirradiated DNA. The fraction of DNA repaired in this fashion at different doses agreed closely with the fraction of cells that survived gamma irradiation with the same doses. Krisch et al. (13) also give direct evidence of the repair of broken DNA on neutral sucrose gradients, using the same strain of E. coli K-12 (AB2497), but using decay of '25I incorporated into the DNA to produce the double-strand breaks. Their results indicate that the WT strain could repair up to three or four double-strand breaks per genome, whereas no repair was observed for the recA13 strain. Krasin and Hutchinson (12) have refined the traditional method of assessing repair of doublestrand breaks to the point where they could measure breakage at gamma ray doses of less than 13 krads. Using this technique, they were able to demonstrate repair of double-strand
J. BACTrERIOL.
breaks during post-irradiation incubation of WT cells (AB2497) but not of recA (AB2487) or recB (NH4803) strains. When the WT strain was grown in aspartate medium, however, as opposed to K-medium, such repair was not observed. They proposed that this was due to the fact that the aspartate-grown cells have only 1.3 genome equivalents of DNA per cell and therefore lack the necessary additional replicas necessary for recombination, while the K-medium cells contain four to five genome equivalents per cell. In our experiments, the glucose-grown cultures had calculated DNA contents of two genome equivalents per cell, which may be the minimum necessary for repair of double-strand breaks. Finally, the three positive indications of double-strand break repair in E. coli have all been made with the same strain, strain AB2497 (12, 13; this paper). Youngs and Smith (18) have reported that this strain (an AB1157 derivative) is more radiation resistant than the derivatives of strain W3110 that they have investigated. This may partially explain their inability to demonstrate double-strand break repair (2). It thus appears that E. coli K-12 AB2497 is indeed capable of repairing several doublestrand breaks per genome by a pathway involving recombination. ACKNOWLEDGMENTS This work was supported by grant DAAG-29-76-C-0034 from the United States Army Research Office and by Public Health Service grant 2-POl-ES00597 from the National Institute of Environmental Health Sciences. K.M.U. was supported by a National Science Foundation National Needs Traineeship.
LITERATURE CITED 1. Bonura, T., and K. C. Smith. 1976. The involvement of indirect effects in cell-killing and DNA double-strand breakage in y-irradiated Escherichia coli K-12. Int. J. Radiat. Biol. 29:293-296. 2. Bonura, T., C. D. Town, K. C. Smith, and H. S. Kaplan. 1975. The influence of oxygen on the yield of DNA double-strand breaks in X-irradiated Escherichia coli K-12. Radiat. Res. 63:567-577. 3. Burrell, A. D., and C. J. Dean. 1975. Repair of doublestrand breaks in Micrococcus radiodurans, p. 507-512. In P. C. Hanawalt and R. B. Setlow (ed.), Molecular mechanisms for repair of DNA, Plenum Press, New York. 4. Burrell, A. D., P. Feldschreiber, and C. J. Dean. 1971. DNA-membrane association and the repair of double breaks in X-irradiated Micrococcus radiodurans. Biochim. Biophys. Acta 247:38-53. 5. Davies, R., and A. J. Sinskey. 1973. Radiation-resistant mutants of Salmonella typhimurium LT2: development and characterization. J. Bacteriol. 113:133-144. 6. Davies, R., A. J. Sinskey, and D. Botstein. 1973. Deoxyribonucleic acid repair in a highly radiation-resistant strain of Salmonella typhimurium. J. Bacteriol. 114: 357-366.
VOL. 138, 1979 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. Kaplan, H. S. 1966. DNA-strand scission and loss of viability after X-irradiation of normal and sensitized bacterial cells. Proc. Natl. Acad. Sci. U.S.A. 55:14421446. 9. Kitayama, S., and A. Matsuyama. 1968. Possibility of the repair of double-strand scissions in Micrococcus radiodurans DNA caused by gamma-rays. Biochem. Biophys. Res. Commun. 33:418422. 10. Kitayama, S., and A. Matsuyama. 1971. Double-strand scissions in DNA of gamma-irradiated Micrococcus radiodurans and their repair during postirradiation in.cubation. Agric. Biol. Chem. 36:644-652. 11. Korch, C., S. 0verbo, and K. Kleppe. 1976. Envelopeassociated folded chromosomes from Escherichia coli: variations under different physiological conditions. J. Bacteriol. 127:904-916. 12. Krasin, F., and F. Hutchinson. 1977. Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. J. Mol. Biol. 116:81-98.
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13. Krisch, R. E., F. Krasin, and C. J. Sauri. 1976. DNA breakage, repair and lethality after '25I decay in rec+ and recA strains of Escherichia coli. Int. J. Radiat.
Biol. 29:37-50. 14. Levin, D., and F. Hutchinson. 1973. Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation. J. Mol. Biol. 75:455-478. 15. Lyderson, D. E., and D. E. Pettijohn. 1977. Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation. Chromosoma 62:199-215. 16. Pettijohn, D. E. 1977. Prokaryotic DNA in nucleoid structure. Crit. Rev. Biochem. 4:175-202. 17. Ulmer, K. M., R. F. Gomez, and A. J. Sinskey. 1979. Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid. J. Bacteriol. 138:475-485. 18. Youngs, D. A., and K. C. Smith. 1976. Single-strand breaks in the DEA of the uvrA and uvrB strains of Escherichia coli K-12 after ultraviolet irradiation. Photochem. Photobiol. 24:533-541.
Vol. 138, No. 2
Ionizing Radiation Damage to the Folded Chromosome of Escherichia coli K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid KEVIN M. ULMER,t* REINALDO F. GOMEZ,' AND ANTHONY J. SINSKEY' Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139' and Joint Program in Biological Oceanography, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 7 September 1978
'The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475-485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of doublestrand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.
Until recently, the available evidence had supported the belief that ionizing radiation-induced DNA double-strand breaks in Escherichia coli are lethal lesions not subject to repair (1, 8). Direct evidence of repair of double-strand breaks has been reported for more radiationresistant bacteria, however. Significant amounts of repair occur in Micrococcus radiodurans (3, 4, 9, 10) and in a highly radiation-resistant strain of Salmonella typhimurium LT2 developed by Davies and Sinskey (5, 6; D. Baraldi and A. J. Sinskey, unpublished data). The detailed mechanisms of repair in these strains have not been elucidated, however, largely due to the lack of suitable, well-characterized repair-deficient mutants. These earlier studies were also plagued by rotor speed effects (14) and by problems associt Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139.
ated with cell lysis and DNA extraction. Shearing during the lysis procedure was unavoidable, and the largest pieces of unirradiated DNA that could be obtained routinely represented only a fraction of the molecular weight of the intact genome (2). Recent technical improvements in these areas have enabled two groups to demonstrate doublestrand break repair in E. coli. Krisch et al. (13) produced DNA double-strand breaks by incorporating 125I into the DNA and allowing for decay. Incubation of damaged cells in growth medium resulted in repair of up to three or four double-strand breaks per genome in the wildtype (WT) strain (strain AB2497), whereas no repair was observed in a recA13 strain (strain AB2487). There was a one-to-one correlation between cell killing and the number of postincubation residual double-strand breaks. With the same E. coli strains, Krasin and Hutchinson
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(12) have obtained quite similar results by using gamma irradiation in an oxygenated solution to produce the double4strand breaks. By using very low gamma ray doses, they have demonstrated that double-strand break repair requires the presence of multiple genome equivalents of DNA per cell as well as functional recA and recB genes. In the present study, using procedures for the gentle isolation and unfolding of nucleoid DNA (17), we demonstrate directly rec-dependent repair of several double-breaks per genome and present evidence for recombinational intermediates involved in this repair process. MATERIALS AND METHODS The procedures for cell growth, labeling, irradiation, nucleoid isolation and unfolding, and sedimentation analysis have been presented in detail in another paper (17). Survival after gamma irradiation. E. coli strains AB2497 WT and AB2487 recA13 were grown overnight in M9 minimal medium. Fresh 20-ml cultures were inoculated with 0.2 ml of the overnight cultures, grown with shaking at 37°C for 4.5 to 5 h, and then quickly cooled in ice water and harvested by centrifugation at 7,500 rpm for 5 min. The cells were washed with 20 ml of M9 salts plus 10,ug of thymine per ml, centrifuged a second time, and finally resuspended in 12 ml of M9 salts plus thymine. Samples were held on ice in glass test tubes (12 by 75 mm) during irradiation. Dilutions were made in phosphate buffer, and the cells were plated in duplicate (0.1 ml/ plate) on M9 minimal medium plates (15 g of agar [Difco] per liter). These were incubated at 37°C for 48 h before colonies were counted.
RESULTS Survival curves for gamma-irradiated E. coli K-12 strains. Gamma irradiation of exponential-phase cells of strains AB2497 WT and AB2487 recA13 at 00C in M9 buffer under aerobic conditions resulted in the survival patterns indicated in Fig. 1. The points shown are the averages of the results of three separate experiments and indicate that for the WT strain, there is an exponential decrease in survival for doses up to 100 krad. The markedly enhanced sensitivity of the recA13 strain irradiated under identical conditions is also apparent. Repair of gamma radiation damage: folded chromosomes. When strain AB2497
WT was incubated at 370C in complete M9 medium after gamma irradiation in M9 buffer at 0°C, major changes occurred in the gradient profiles of folded chromosomes. Figure 2 demonstrates the effects of post-irradiation incubation after a dose of 10 krad. Immediately after irradiation, the nucleoids were shifted toward the top of the gradient, and the sedimentation
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FIG. 1. Survival of E. coli strains AB2497 WT on minimal medium plates after gamma irradiation in M9 buffer.
(0) and AB2487 recA13 (0)
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FIG. 2. Gradient profiles for repair of gamma-irradiated (10 krads) folded chromosomes of E. coli strain AB2497 WT. Gradients were centrifuged at 2,949 rpm, for a total integrated centrifugal force (42t) of 6.00 x 109 rad'/s. (A) Symbols: 0, pre-irradiation; 0, immediately post-irradiation; A, 0.5-h incubation. (B) Symbols: 0, 1-h incubation; 0, 2-h incubation; A, 3-h incubation.
coefficient was reduced from 1,762S to only 1,370S. After 0.5 h in growth medium, the entire peak shifted to 2,050S, which is in the range of the most rapidly sedimenting nucleoids in the original, unirradiated profile. The sedimentation rate continued to increase, reaching 2,318S after 1 h. The material was more heterogeneous than that of the unirradiated profile, and the peak
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was broadened accordingly. After 2 h of incubation, the peak sharpened more, and the material moved back toward the top of the gradient to a position at the low-S side of the unirradiated distribution (1,686S). Finally, after 3 h, the nucleoids once again shifted slightly toward more rapidly sedimenting material, returning to the sedimentation rate of unirradiated nucleoids (1,793S). With increasing length of incubation, increasing amounts of rapidly sedimenting material appeared in the last few fractions at the bottom of the gradients as well. Any material in the last fraction at the bottom of the gradient had a sedimentation coefficient greater than 3,100S, which is the range observed for membrane-associated nucleoids (15, 16). When strain AB2487 recA13 was incubated at 370C in complete M9 medium after receiving a dose of 5 krad in M9 buffer at 00C, the gradient profiles for nucleoids were significantly different from those observed with the WT strain (Fig. 3). Immediately after irradiation, the peak shifted toward the top of the gradient, as was observed previously, but upon incubation the sedimentation coefficients of the material continued to decrease until most of the material was at the very top of the gradient. No rapidly sedimenting material was formed during incubation, as revealed by the total absence of radioactivity below the position of the unirradiated peak in the 10 to 50% gradients used for this experiment. A high-density shelf was included in these gradients as well, to detect any very rapidly sedimenting material that might be lost to the bottom of the tube. The only indication of possible repair was the presence of a small shoulder on
J. BACTERIOL.
the 3-h incubation profile at the right-hand side of the initial, unirradiated peak. Repair of gamma radiation damage: RNase-unfolded chromosomes. When nucleoids from AB2497 WT were unfolded by digestion with RNase after post-irradiation incubation in M9 complete medium, gradient profiles like those in Fig. 4 were obtained. A large decrease in the sedimentation rate was observed immediately after irradiation. The sedimentation coefficient for the unirradiated nucleoids was 1,098S, which was reduced to only 786S after a 5-krad irradiation. Return ofthis material to the position of unirradiated material in the gradients was quite rapid, as indicated by the 0.5-h incubation profile (1,042S). This material was fairly heterogeneous, and included a range of sedimentation coefficients on either side of the unirradiated nucleoid peak. With further incubation, the profile sharpened, and by 3 h the width was the same as that of the profile of unirradiated chromosomes. The mean sedimentation coefficient after 3 h of incubation (1,133S) was slightly increased over the value for unirradiated DNA. The extent of repair after 2 h of post-irradiation incubation at several doses of gamma rays
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8 6 4
25
2 2
0.
E 5,
A
0.4 0.2 0.6 0.8 FRACTIONAL DISTANCE SEDIMENTED
FIG. 3. Gradient profiles for repair of gamma-irradiated (5 krads) folded chromosomes from E. coli strain AB2487 recA13. Gradients were centrifuged at an average rotor speed of 2,781 rpm, for a total integrated centrifugal force (w2t) of 6.00 x l0o rad2/s. Symbols: v- *, pre-irradiation; 0-0, immediately post-irradiation; A A, 0.5-h incubation; v X, 3-h incubation. -
0.8 0.4 0.6 0.2 FRACTIONAL DISTANCE SEDIMENTED
FIG. 4. Gradient profiles for repair of RNase-unfolded chromosomes of E. coli strain AB2497 WT after a 5-krad gamma irradiation. The gradients were centrifuged at an average rotor speed of about 3,000 rpm, for a total integrated centrifugal force (W2t) of 9.00 x 109 rad2/s. (A) Symbols: 0, unirradiated; 0, immediately post-irradiation; A, 0.5-h incubation. (B) Symbols: *, 1-h incubation; 0, 2-h incubation; A, 3-h incubation.
ESCHERICHIA CHROMOSOME REPAIR
'VOL. 138, 1979 determined in a final experiment (Fig. 5). Complete recovery occurred after doses of 0.5 to 2.0 krads within this 2-h incubation period, whereas some material with lower sedimentation coefficients was still present in the profiles for 5.0 and 10.0 krads. This is consistent with the results shown in Fig. 4, where 3 h of incubation was required before all of the low-S material had been repaired. That fraction of the material that did not return to the position for unirradiated chromosomes appeared to sediment to the very bottom of the gradient, although some small amount of material also appeared at the top of the 5- and 10-krad profiles. The percentage of material that did return to the position of unirradiated chromosomes is indicated in Table 1, along with the percent survival at the same doses from Fig. 1. The agreement between the two sets of numbers is quite good, suggesting that the percentage of RNase-unfolded chromosomes repaired was equivalent to the percentage of cells that survived.
was
DISCUSSION Repair of gamma radiation damage to folded chromosomes. When E. coli WT cells were incubated for 2 to 3 h in complete growth medium at 370C after gamma irradiation in buffer at 0°C under aerobic conditions, significant amounts of material returned to the posiIC0
A
2 6
4
2
a. B
FRACTIONAL DISTANCE SEDIMENTED
FIG. 5. Gradient profiles for 2-h repair of RNaseunfolded chromosomes of E. coli strain AB2497 WT after several doses of gamma rays. Centrifugation was carried out at an average rotor speed of 3,584 rpm, for a total integrated centrifugal force (w2t) of 9.00 x 109 rad2/s. (A) Symbols: 0, unirradiated; 0, 0.5 krad; A, 1.0 krad. (B) Symbols: 0, 2.0 krads; 0, 5.0 krads; A, 10.0 krads.
489
TABLE 1. Recovery of RNase-digested nucleoids after 2 h of post-irradiation incubation, and cell survival Dose (krad)
% nucleoids' Recovery of
% Survivalb
0.5 1.0 2.0 5.0 10.0
98 93 81 79 41
95 91 83 63 40
a From Fig. 5. The major peaks obtained after 2 h of repair were integrated and expressed as a percentage of the integrated peak for unirradiated chromozomes. b From Fig. 1.
tion of unirradiated nucleoids in the gradients. This behavior was not observed after irradiation of a recA13 strain, suggesting that repair of folded chromosomes may require recombination. The 2,300S peak observed after the fit hour of incubation may represent a recombinational intermediate, since it is in the range of sedimentation coefficients of nearly replicated, double chromosomes (11). The kinetics of its appearance rule out the possibility that direct, radiation-induced cross-linking is responsible for its formation. Subsequent shifts in the profile may indicate segregation of daughter chromosomes before the onset of normal replication and division. Several researchers have reported the formation of rapidly sedimenting, membrane-associated material during repair of ionizing radiation damage (3, 4; Baraldi and Sinskey, unpublished data). In the present experiments, during the later stages of post-irradiation incubation, large amounts of material with sedimentation coefficients in excess of 3,100S were formed. This is within the range of sedimentation rates observed for membrane-associated nucleoids (16) and suggests that radiation-induced cell death may result from the inability of cells to properly segregate daughter chromosomes from the membrane (7). Repair of double-strand breaks. The problem that has confronted all previous investigations of repair of double-strand breaks in E. coli has been to measure viability and the rate of formation of double-strand breaks in the same biologically significant dose range. Unfortunately, this has generally not been possible. The background level of double-strand breaks accumulated during cell lysis precluded measurement of repair of less than six or seven doublestrand breaks per genome (1, 8). In an attempt to circumvent this shortcoming, the number of breaks expected in the biologically significant dose range was estimated by extrapolation from
490
ULMER, GOMEZ, AND SINSKEY
measurements of breakage at higher doses and compared with the number of lethal hits determined from survival curves at the lower doses. The most recent calculations of this type indicate that 1.3 to 1.4 double-strand breaks occur per lethal event (2). Our procedure for isolating nucleoids yielded minimally damaged DNA. Few, if any, singlestrand breaks occurred during isolation, as evidenced by the presence of supercoiling, and when nucleoids were examined under an electron microscope, no free ends were observed, indicating that no double-strand breaks were formed. The gentle unfolding techniques that we have developed (RNase or heat) yielded DNA of very high molecular weight, essentially eliminating the problem of background breaks and permitting direct measurement of doublestrand break formation by gamma rays in the biologically significant dose range of less than 10 krads. An average of one lethal event per cell corresponds to a survival of 37%, which occurs at a dose of 10 krads for strain AB2497 (Fig. 1). The average number of double-strand breaks per genome at this dose is 2.7, based on data from our experiments (17), or 3.7, based on the data of Lyderson and Pettijohn (15) for in vivo gamma irradiation of nucleoids. The absolute values of these figures are still subject to question, however, due to uncertainties surrounding the initial conformation of the DNA, and these conclusions can only be considered suggestive of double-strand break repair. The more conclusive evidence of doublestrand break repair comes from experiments reported here, in which post-irradiation incubation of the WT cells resulted in the return of initially broken, RNase-unfolded chromosomal DNA to the same position in the gradient as that of unirradiated DNA. The fraction of DNA repaired in this fashion at different doses agreed closely with the fraction of cells that survived gamma irradiation with the same doses. Krisch et al. (13) also give direct evidence of the repair of broken DNA on neutral sucrose gradients, using the same strain of E. coli K-12 (AB2497), but using decay of '25I incorporated into the DNA to produce the double-strand breaks. Their results indicate that the WT strain could repair up to three or four double-strand breaks per genome, whereas no repair was observed for the recA13 strain. Krasin and Hutchinson (12) have refined the traditional method of assessing repair of doublestrand breaks to the point where they could measure breakage at gamma ray doses of less than 13 krads. Using this technique, they were able to demonstrate repair of double-strand
J. BACTrERIOL.
breaks during post-irradiation incubation of WT cells (AB2497) but not of recA (AB2487) or recB (NH4803) strains. When the WT strain was grown in aspartate medium, however, as opposed to K-medium, such repair was not observed. They proposed that this was due to the fact that the aspartate-grown cells have only 1.3 genome equivalents of DNA per cell and therefore lack the necessary additional replicas necessary for recombination, while the K-medium cells contain four to five genome equivalents per cell. In our experiments, the glucose-grown cultures had calculated DNA contents of two genome equivalents per cell, which may be the minimum necessary for repair of double-strand breaks. Finally, the three positive indications of double-strand break repair in E. coli have all been made with the same strain, strain AB2497 (12, 13; this paper). Youngs and Smith (18) have reported that this strain (an AB1157 derivative) is more radiation resistant than the derivatives of strain W3110 that they have investigated. This may partially explain their inability to demonstrate double-strand break repair (2). It thus appears that E. coli K-12 AB2497 is indeed capable of repairing several doublestrand breaks per genome by a pathway involving recombination. ACKNOWLEDGMENTS This work was supported by grant DAAG-29-76-C-0034 from the United States Army Research Office and by Public Health Service grant 2-POl-ES00597 from the National Institute of Environmental Health Sciences. K.M.U. was supported by a National Science Foundation National Needs Traineeship.
LITERATURE CITED 1. Bonura, T., and K. C. Smith. 1976. The involvement of indirect effects in cell-killing and DNA double-strand breakage in y-irradiated Escherichia coli K-12. Int. J. Radiat. Biol. 29:293-296. 2. Bonura, T., C. D. Town, K. C. Smith, and H. S. Kaplan. 1975. The influence of oxygen on the yield of DNA double-strand breaks in X-irradiated Escherichia coli K-12. Radiat. Res. 63:567-577. 3. Burrell, A. D., and C. J. Dean. 1975. Repair of doublestrand breaks in Micrococcus radiodurans, p. 507-512. In P. C. Hanawalt and R. B. Setlow (ed.), Molecular mechanisms for repair of DNA, Plenum Press, New York. 4. Burrell, A. D., P. Feldschreiber, and C. J. Dean. 1971. DNA-membrane association and the repair of double breaks in X-irradiated Micrococcus radiodurans. Biochim. Biophys. Acta 247:38-53. 5. Davies, R., and A. J. Sinskey. 1973. Radiation-resistant mutants of Salmonella typhimurium LT2: development and characterization. J. Bacteriol. 113:133-144. 6. Davies, R., A. J. Sinskey, and D. Botstein. 1973. Deoxyribonucleic acid repair in a highly radiation-resistant strain of Salmonella typhimurium. J. Bacteriol. 114: 357-366.
VOL. 138, 1979 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. Kaplan, H. S. 1966. DNA-strand scission and loss of viability after X-irradiation of normal and sensitized bacterial cells. Proc. Natl. Acad. Sci. U.S.A. 55:14421446. 9. Kitayama, S., and A. Matsuyama. 1968. Possibility of the repair of double-strand scissions in Micrococcus radiodurans DNA caused by gamma-rays. Biochem. Biophys. Res. Commun. 33:418422. 10. Kitayama, S., and A. Matsuyama. 1971. Double-strand scissions in DNA of gamma-irradiated Micrococcus radiodurans and their repair during postirradiation in.cubation. Agric. Biol. Chem. 36:644-652. 11. Korch, C., S. 0verbo, and K. Kleppe. 1976. Envelopeassociated folded chromosomes from Escherichia coli: variations under different physiological conditions. J. Bacteriol. 127:904-916. 12. Krasin, F., and F. Hutchinson. 1977. Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. J. Mol. Biol. 116:81-98.
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13. Krisch, R. E., F. Krasin, and C. J. Sauri. 1976. DNA breakage, repair and lethality after '25I decay in rec+ and recA strains of Escherichia coli. Int. J. Radiat.
Biol. 29:37-50. 14. Levin, D., and F. Hutchinson. 1973. Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation. J. Mol. Biol. 75:455-478. 15. Lyderson, D. E., and D. E. Pettijohn. 1977. Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation. Chromosoma 62:199-215. 16. Pettijohn, D. E. 1977. Prokaryotic DNA in nucleoid structure. Crit. Rev. Biochem. 4:175-202. 17. Ulmer, K. M., R. F. Gomez, and A. J. Sinskey. 1979. Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid. J. Bacteriol. 138:475-485. 18. Youngs, D. A., and K. C. Smith. 1976. Single-strand breaks in the DEA of the uvrA and uvrB strains of Escherichia coli K-12 after ultraviolet irradiation. Photochem. Photobiol. 24:533-541.
