Vol. 130, No. 1 Printed in U.S.A.
JOURNAL OF BACTEROLOGY, Apr. 1977, p. 187-191 Copyright 0 1977 American Society for Microbiology
Repair of Hydrogen Peroxide-Induced Single-Strand Breaks in Escherichia coli Deoxyribonucleic Acid H. N. ANANTHASWAMY1 AND A. EISENSTARK* Division of Biological Sciences, University of Missouri, Columbia, Missouri 65201
Received for publication 19 October 1976
Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H202 (a toxic photoproduct) that was selectively lethal for rec and polAl Escherichia coli mutants. H202 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polAl, recA recB, and recA mutants, but were efficiently repaired in wildtype strains. We conclude that H202 deoxyribonucleic acid lesions require both the polA + and recA + pathways for repair.
Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan in the presence of oxygen yields a photoproduct that kills bacteria (23). The toxic component of the tryptophan photoproduct has been identified as hydrogen peroxide (H202) (14). We reported previously that both this toxic component and H202 exert identical biological effects on phage deoxyribonucleic acid (DNA) (2). Both the photoproduct and H202 act synergistically with near-ultraviolet radiation to kill cells and to enhance singlestrand (SS) DNA breaks, as well as to kill T7 phage (2). The effect of H202 on isolated DNA has been studied by several workers (7, 16, 21, 22) who found that H202 alters DNA to liberate all four bases; also, ultraviolet absorption and melting temperatures are decreased (16, 21, 22). Apparently, H202 breaks the sugar-phosphate backbone sufficiently to inactivate transforming DNA (7). However, most of these studies were conducted on isolated DNA using relatively high concentrations of H202 (0.05 to 0.1 M) and a long period of incubation in the presence of FeCl3. Whereas X-ray-induced SS DNA breaks may be repaired (11, 15, 19, 20) (and X rays are known to produce H202), the repair of H202induced SS breaks has not been reported previ-
The present paper describes H202 induction of SS breaks in DNA and the ability of various mutants to repair these breaks. The results indicate that H202-induced SS breaks in the DNA can be repaired in wild-type E. coli by processes similar to those described for the repair of X-ray-induced SS breaks in E. coli (20). It is concluded that the increased sensitivity of the polAl, recA recB, and recA strains to H202 is due to their reduced capacity to repair the SS breaks induced by H202.
MATERIALS AND METHODS Bacterial strains and growth. The strains listed in Table 1 were used in the experiments described below. Bacteria were routinely grown at 37°C with aeration in M9 medium (3) containing glucose (2 g/ liter) and Casamino Acids (2 mg/ml) and supplemented with required ingredients (thymine, 10 ,ug/ ml; thiamine, 1 ,ig/ml; and threonine, isoleucine, and valine, all 10 ,ug/ml). Survival curves. All strains were grown to about 108 cells/ml and harvested by centrifugation at 40C. The cells were washed once with 0.05 M potassium phosphate buffer (pH 7.4) and resuspended in the same buffer at room temperature (250C) to one-tenth the original volume. A 1:10 dilution of the cell suspension was made in either phosphate buffer or phosphate buffer containing H202 (final concentration, 0.01 M) and incubated at 25°C. At various time ously. samples were withdrawn, diluted immeTwo distinct types of repair processes, desig- intervals,and plated in duplicate on nutrient agar. nated as types II and III, operate inEscherichia diately, Plates were incubated for 24 to 48 h at 37°C for coli for the rejoining of X-ray-induced SS colony formation. breaks (20). The type II repair process, which Alkaline sucrose gradient studies. The induction takes place in buffer at room temperature (15 to of SS breaks by H202 followed by the ability to repair 20 min), requires functional polymerase I activ- these breaks in various bacterial strains was studied ity (20). In contrast, the type III repair process by sedimentation through alkaline sucrose graof the procedure of requires full growth medium conditions at 370C dients by a slight modification McGrath and Williams (15). Cells were labeled with and functional rec gene products (20). 20 yCi of [3H]thymidine per ml (specific activity, 45 ' Present address: Division of Medical Physics, Univer- Ci/mmol; New England Nuclear Corp., Boston, Mass.) for at least three generations, harvested by sity of California, Berkeley, CA 94720. 187
188
ANANTHASWAMY AND EISENSTARK
J. BACTERIOL.
TABLE 1. Strains used Designation
W3110 P3478 JC5029 JC5088 KL168 X9247
Relevant genotypea
polAl
recA56 recB21 recA56 recB21
Other markers
thy thy thi-1 thi-1 thi-1 thi-1
Reference or source
ilv-318 thr-300 rel-1 spc-300 Ailv-318 thr-300 rel-1 spc-300 Xdrm-3 rel-1 Xilv-318 thr-300 spc-300 X-
6 6 5 5 B. J. Bachmann Argonne National
Laboratory W3110 and P3478 are isogenic. JC5029, JC5088, and X9247 are isogenic. KL168 is closely related to JC5029 and JC5088. a
centrifugation, and treated with 0.01 M H202 as described above. After a 10-min treatment at 25°C, 10,ug of catalase per ml (Sigma Chemical Co., St. Louis, Mo.) was added to destroy H202, and a portion was removed quickly and stored in ice at 0°C. The remaining suspension was allowed to stand at room temperature for 15 min, after which time another portion including an untreated control (kept in ice) were gently layered on top of 0.2 ml of lysing solumedium (M9-glucose-Casamino Acids supplemented with the required ingredients) and incubated at 37°C for 40 min. At the end, 0.05-ml samples from each portion including and untreated control (kept in ice) were gently layered on top of 0.2 ml of lysing solution (0.5 M NaOH and 0.05% sodium dodecyl sulfate), which was already layered on a preformed (4.8 ml) 5 to 20% alkaline sucrose gradient (0.5 M NaCl, 0.2 M NaOH and 0.01 M ethylenediaminetetraacetic acid). After standing for 30 min at room temperature for lysis, the gradients were centrifuged at 30,000 rpm for 90 min at 20°C, using an SW50.1 rotor in a Beckman L5-50 ultracentrifuge. Six-drop fractions were collected from the bottom of the tube onto 3MM filter paper disks (Whatman; 2.3 cm) and washed once with 5% trichloroacetic acid and twice with 95% ethanol at room temperature. The dried disks were then placed in vials with about 6 ml of Liquifluor (New England Nuclear Corp.) and counted in a Beckman liquid scintillation counter. The number-average molecular weight (Mn) was calculated, using T7 phage DNA as a marker, according to the formula described by Ley (12).
RESULTS Effect of H202 on survival of bacteria. H202 survival curves of the six strains tested are shown in Fig. 1. The polymerase I-deficient strain P3478 polAl and the X9247 recA recB double mutant were extremely sensitive to H202 compared with the other strains ofE. coli. However, JC5088 recA exhibited increased sensitivity to H202 only in the first log of killing. In contrast, the KL168 recB strain was less sensitive to H202 than the recA strain. Both of the wild-type strains (JC5029 and W3110) exhibited broad shoulders compared with the single-hit kinetics of the mutant strains. An identical control of each strain kept in phosphate buffer alone (no H202) during the entire experiment
z
0
a: c0 0
z
5)
TIME (MINUTES)
FIG. 1. Sensitivity of various E. coli strains to H202. Cells in exponential phase were exposed to 0.01 M H202 in phosphate buffer at room temperature (25°C). JC5029 wild type; W3110 wild type; KL168 recB; JC5088 recA; X9247 recArecB; P3478 polAl.
did not show any loss in colony-forming ability (data not shown). The different sensitivities of the various strains to H202 could be due to the relative differences in their ability to repair the lesions induced by H202. Induction and repair of SS breaks. The existence of two distinct types of repair mechanisms for the repair of SS breaks induced in E. coli after aerobic irradiation with X rays (20), plus our own observation that polAl and rec mutants are considerably more sensitive to H202 than wild-type E. coli, suggested that similar mechanisms might be involved in the repair of
VOL. 130, 1977
REPAIR OF H202-INDUCED BREAKS IN E. COLI DNA
H202-induced SS breaks in the DNA. To demonstrate this, the procedure described by Town et al. (19) was used (see Materials and Methods) to examine polAl-mediated repair (buffer repair or type II repair) and rec-dependent repair (medium-dependent, type Ill repair) processes. Although we are referring to the polAl dependent buffer repair as type II repair and rec-dependent medium repair as type III repair, no distinction has been made to exclude type II and III repairs mediated by polymerase III (9). Recently, Hamelin et al. (9) have shown that DNA polymerase III is involved in type II (in polAl cells) and type III (in polA + and polAl) repairs of gamma-ray-induced DNA SS breaks. The number of SS breaks induced by H202, as well as the number of SS breaks remaining after type II and type III repairs, was calculated from the following equation: number of SS breaks/25 x 108 daltons of DNA -
=25
x
(1
1n
Mnt Mn
where Mnt equals the number-average molecular weight of the treated DNA and Mn0 equals the number-average molecular weight of the untreated control DNA. The induction of SS breaks by H202, followed by the ability to repair these breaks by type II 5.0
W3110
4.0
C'
zA
Z
£
3.0
2.0
1.0
.0 1.0
0.8 FRACTIONAL
0.6
DI STANCE
0.4 0.2 SEDI MENTED
0
FIG. 2. Alkaline sucrose gradient profiles of DNA from E. coli W3110 cells after various treatments. (0) Untreated control (Mn0 = 37 x 10"); (@) 10 min, H202, no incubation (Mnt = 11 x 106); (A) 10 min, H202, 15-min incubation in buffer at 25°C (Mnt = 18 x 106); (A) 10 min, H202, 15-min incubation in buffer at 25°C, 40-min incubation at 370C in full growth medium (Mnt = 30 x 106). The numberaverage molecular weights (Mn) were calculated as described by Ley (12). The direction of sedimentation is from right to left.
189
TABLE 2. Repair of H202-induced SS breaks SS breaks/E. coli genomea Strain
W3110 (wild type) P3478 (polAl) X9247 (recA recB) JC5088 (recA) KL168 (recB) JC5029 (wild type) a
After Aftr AteAr type H0 tyeII and III
treatment II 153 74
239 221 178 148 120
230 182 102 112 51
repairsc
14 154 127 54 37 36
Calculated for each strain from their respective
Mn. and Mn, values mentioned in the legend to Fig. 2 according to the equation given in the text. b In buffer at 25°C for 15 min (requires polA+). c In broth at 37°C for 40 min (requires rec+).
and type III processes in wild-type W3110 and mutant strains, can be inferred from the sedimentation profiles presented in Fig. 2. In addition, calculations of the number of SS breaks per E. coli genome (25 x 108 daltons) (4) after various treatment for each strain (Table 2) clearly indicate the relative differences among various strains in their ability to repair the breaks induced by H202. Brief treatment (10 min) of cells with H202 resulted in the induction of SS breaks to varying degrees, depending on the strain (Table 2). The relative differences in the initial number of observable SS breaks induced by H202 between various strains could be due to one or both of two factors. (i) They could be due to differences in the ability of the various strains to rejoin the SS break rapidly during the 10-min H202 treatment period at 250C. Thus, in repair-proficient strains a certain niumber of SS breaks might have already been repaired by the end of the H202 treatment period. (ii) They could be due to differences in the level of intracellular catalase activity between various strains such that the actual amount of H202 available to react with the DNA in each strain might be different. Although the level of endogenous catalase activity in cell extracts of all the strains has not been measured, at least in two cases (strains JC5088 recA and P3478 polAl) no differences in the level of catalase activity were observed compared with their respective isogenic wild-type parents (JC5029 and W3110) (data not shown). In addition, no significant differences in the level of catalase activity were observed between two wild-type strains (W3110 and JC5029). In this connection, Adler (1) has reported that there is no correlation between the level of catalase activity and sensitivity of E. coli to reagent H202. One of his explanations for the inability of catalase to protect cells
190 ANANTHASWAMY AND EISENSTARK against added H202 is that high concentrations of H202 may inactivate the catalase in cells. Therefore, based on these observations, we can infer that the differences in the initial number of observable SS breaks after peroxide treatment are due to the differences in the ability of the various strains to rejoin the SS breaks during the treatment period. In fact, Town et al. (19) have reported similar differences between strains W3110 pol+ and P3478 polAl in the yield of SS break for a given dose of X rays when irradiated at room temperature but not at OOC. Nevertheless, the relative differences in the ability of the various strains to rejoin the SS breaks induced by H202 become apparent when the number of SS breaks remaining unrepaired after completion of type II and type III repairs is taken into consideration. In the case of P3478 polAl and X9247 recA recB strains, the number of SS breaks remaining unrepaired is significantly higher (154 and 127, respectively) than in the other strains (14 and 54, respectively) (Table 2). In other words, about 60 to 70% of the initial yield of SS breaks is not repaired in these two strains compared with only about 9 to 30% unrepaired breaks in other strains. However, these calculations are only relative and are not strictly accurate because (i) of the differences in the initial yield of SS breaks for reasons discussed above and (ii) the Mn value calculation is extremely sensitive to the small amount of DNA in the upper portions of the gradient (11). DISCUSSION The H202 induction of SS breaks in the DNA (upon treatment of bacteria) supports earlier studies of H202 damage to isolated DNA. The increased sensitivity of polAl, recA recB, and recA strains to H202 compared with the other strains suggests that the lesions induced by H202 are probably not repaired in these mutant strains and that the unrepaired breaks might be the cause for cell death. Similar increased sensitivity of polAl, recA recB, and recA mutants to X rays has been attributed to the inability of these strains to repair the SS breaks induced by X rays (11, 19). Apart from strand breakage, other damages to the DNA such as damages to base and sugar moieties have been thought to be involved in radiation-induced lethality (8, 10, 13, 17, 18). Since H202 is also known to liberate all four bases from the DNA in addition to the breakage of the sugar-phosphate backbone (16), it is quite possible that damages other than strand breakage might also contribute to lethality in bacteria exposed to H202. However, the results of the alkaline sucrose
J. BACTERIOL.
gradient studies (Table 2) reveal that even brief treatment (10 min) of bacteria with low concentrations of H202 (0.01 M) results in the induction of a substantial number of SS breaks. The observed differences in the initial yield of SS breaks by H202 between various strains could be attributed to their relative differences in their ability to rapidly rejoin the breaks during the H202 treatment period. This agrees with the results of Town et al. (19), who reported previously that the differences in the initial yield of SS breaks for a given dose of X rays in W3110 and P3478 polAl strains after aerobic irradiation at room temperature are due to the absence of a rapid repair system in polAl strain, which is present in the wild-type strain. Furthermore, when irradiations were carried out at 0°C, identical yields of SS breaks were obtained in both the strains for a given dose of X rays (19). Analyses of the sucrose gradient sedimentation data indicate that a large number of breaks are left unrepaired after post-treatment incubation in polAl, recA recB, and recA strains compared with the other strains (Table 2). This correlates with the survival data indicating that the increased sensitivities of these mutant strains to H202 are due to their reduced capacity to repair the H202-induced SS breaks. The finding that the recA recB double mutant is comparatively more sensitive to H202 (Fig. 1) and less capable of rejoining the SS breaks than the recA mutant (Table 2) is hard to explain, since the recB mutant exhibits normal capacity to rejoin the H202-induced SS breaks. In contrast, the recA recB double mutant was found to be only slightly more sensitive to X rays than the recA strain, and both strains exhibited more or less identical repair capabilities (11). This discrepancy could be due to differences in the mutant strains used. In summary, we have shown that H202 induces SS breaks in the DNA of bacteria and that these breaks can be repaired by polymerase I-dependent and rec-dependent pathways similar to those already recognized for the repair of X-ray-induced SS breaks in E. coli (20). Furthermore, the results also support the conclusion that the increased sensitivity ofthe mutant strains to H202 is due to their reduced capacity to repair the SS breaks. However, the involvement of polymerases II and III in the repair of H202-induced SS breaks is yet to be ascertained. ACKNOWLEDGMENIS We wish to thank J. Willis for assistance in the calculation of Mn values. This investigation was supported by Public Health Service grant no. 2R01 FD 00 658-05.
VOL. 130, 1977
REPAIR OF H202-INDUCED BREAKS IN E. COLI DNA LITERATURE CITED
1. Adler, H. I. 1963. Catalase, hydrogen peroxide and ionizing radiation. Radiat. Res. Suppl. 3:110-129. 2. Ananthaswamy, H. N., and A. Eisenstark. 1976. NearUV-induced breaks in phage DNA: sensitization by H202 (a tryptophan photoproduct). Photochem. Photobiol. 24:439-442. 3. Anderson, E. H. 1946. Growth requirements of virusresistant mutants of Escherichia coli strain "B." Proc. Natl. Acad. Sci. U.S.A. 32:120-128. 4. Cairns, J. 1963. The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol. 6:208-213. 5. Clark, A. J. 1967. The beginning of a genetic analysis of recombination frequency. J. Cell Physiol. 70(Suppl. 1):165-180. 6. DeLucia, P., and J. Cairns. 1969. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1164-1166. 7. Freese, E. G., J. Gerson, H. Taber, H. J. Rhaese, and E. Freese. 1967. Inactivating DNA alterations induced by peroxides and peroxide-producing agents. Mutat. Res. 4:517-531. 8. Freifelder, D., S. Donta, and R. Goldstein. 1972. X-ray inactivation of bacteriophages: role of 02-dependent damage in single- and double-strand DNA phages. Virology 50:516-519. 9. Hamelin, C., D. A. Youngs, and K. C. Smith. 1976. Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation. J. Bacteriol. 127:1307-1314. 10. Hariharan, P. V., and P. A. Cerutti. 1972. Formation and repair of y-ray-induced thymine damage in Micrococcus radiodurans. J. Mol. Biol. 66:65-81. 11. Kapp, D. S., and K. C. Smith. 1970. Repair of radiationinduced damage in Escherichia coli. H. 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. 12. Ley, R. D. 1973. Post replication repair in an excisiondefective mutant of Escherichia coli: ultra-violet
191
light-induced incorporation of bromodeoxyuridine into parental DNA. Photochem. Photobiol. 18:87-95. 13. Lytle, C. D., and W. Ginoza. 1968. Frequency of singlestrand breaks per lethal y-ray hit in 0X174 DNA. Int. J. Radiat. Biol. 14:553-560. 14. McCormick, J. P., J. R. Fischer, J. P. Pachlatko, and A. Eisenstark. 1976. Characterization of a cell-lethal product from the photooxidation of tryptophan: hydrogen peroxide. Science 191:468-469. 15. 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.
16. Rhaese, H. J., and E. Freese. 1968. Chemical analysis of DNA alterations. I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine. Bio-
chim. Biophys. Acta 155:476-490. 17. Schans, G.P. vander, J. F. Bleichrodt, and J. Blok. 1973. Contribution of various types of damage to inactivation of a biologically-active double-stranded circular DNA by gamma-radiation. Int. J. Radiat. Biol.
23:133-150.
18. Taylor, W., and W. Ginoza. 1967. Correlation of y-ray inactivation and strand scission in the replicative form of 0X174 bacteriophage DNA. Proc. Natl. Acad.
Sci. U.S.A. 58:1753-1757.
19. 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. 20. Town, C. D., K. C. Smith, and H. S. Kaplan. 1973. Repair of X-ray damage to bacterial DNA. Curr. Top.
Radiat. Res. Q. 8:351-399. 21. Uchida, Y., H. Shigematsu, and K. Yamafugi. 1965. The mode of action of hydrogen peroxide on deoxyribonucleic acid. Enzymologia 29:369-376. 22. Yamafugi, K., and Y. Uchida. 1966. Liberation of adenine from deoxyribonucleic acid by hydrogen peroxide. Nature (London) 209:301-302. 23. Yoakum, G., and A. Eisenstark. 1972. Toxicity of Ltryptophan photoproduct on recombinationless (rec) mutants of Salmonella typhimurium. J. Bacteriol. 112:653-655.
JOURNAL OF BACTEROLOGY, Apr. 1977, p. 187-191 Copyright 0 1977 American Society for Microbiology
Repair of Hydrogen Peroxide-Induced Single-Strand Breaks in Escherichia coli Deoxyribonucleic Acid H. N. ANANTHASWAMY1 AND A. EISENSTARK* Division of Biological Sciences, University of Missouri, Columbia, Missouri 65201
Received for publication 19 October 1976
Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H202 (a toxic photoproduct) that was selectively lethal for rec and polAl Escherichia coli mutants. H202 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks. These breaks were repaired to only a small extent in polAl, recA recB, and recA mutants, but were efficiently repaired in wildtype strains. We conclude that H202 deoxyribonucleic acid lesions require both the polA + and recA + pathways for repair.
Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan in the presence of oxygen yields a photoproduct that kills bacteria (23). The toxic component of the tryptophan photoproduct has been identified as hydrogen peroxide (H202) (14). We reported previously that both this toxic component and H202 exert identical biological effects on phage deoxyribonucleic acid (DNA) (2). Both the photoproduct and H202 act synergistically with near-ultraviolet radiation to kill cells and to enhance singlestrand (SS) DNA breaks, as well as to kill T7 phage (2). The effect of H202 on isolated DNA has been studied by several workers (7, 16, 21, 22) who found that H202 alters DNA to liberate all four bases; also, ultraviolet absorption and melting temperatures are decreased (16, 21, 22). Apparently, H202 breaks the sugar-phosphate backbone sufficiently to inactivate transforming DNA (7). However, most of these studies were conducted on isolated DNA using relatively high concentrations of H202 (0.05 to 0.1 M) and a long period of incubation in the presence of FeCl3. Whereas X-ray-induced SS DNA breaks may be repaired (11, 15, 19, 20) (and X rays are known to produce H202), the repair of H202induced SS breaks has not been reported previ-
The present paper describes H202 induction of SS breaks in DNA and the ability of various mutants to repair these breaks. The results indicate that H202-induced SS breaks in the DNA can be repaired in wild-type E. coli by processes similar to those described for the repair of X-ray-induced SS breaks in E. coli (20). It is concluded that the increased sensitivity of the polAl, recA recB, and recA strains to H202 is due to their reduced capacity to repair the SS breaks induced by H202.
MATERIALS AND METHODS Bacterial strains and growth. The strains listed in Table 1 were used in the experiments described below. Bacteria were routinely grown at 37°C with aeration in M9 medium (3) containing glucose (2 g/ liter) and Casamino Acids (2 mg/ml) and supplemented with required ingredients (thymine, 10 ,ug/ ml; thiamine, 1 ,ig/ml; and threonine, isoleucine, and valine, all 10 ,ug/ml). Survival curves. All strains were grown to about 108 cells/ml and harvested by centrifugation at 40C. The cells were washed once with 0.05 M potassium phosphate buffer (pH 7.4) and resuspended in the same buffer at room temperature (250C) to one-tenth the original volume. A 1:10 dilution of the cell suspension was made in either phosphate buffer or phosphate buffer containing H202 (final concentration, 0.01 M) and incubated at 25°C. At various time ously. samples were withdrawn, diluted immeTwo distinct types of repair processes, desig- intervals,and plated in duplicate on nutrient agar. nated as types II and III, operate inEscherichia diately, Plates were incubated for 24 to 48 h at 37°C for coli for the rejoining of X-ray-induced SS colony formation. breaks (20). The type II repair process, which Alkaline sucrose gradient studies. The induction takes place in buffer at room temperature (15 to of SS breaks by H202 followed by the ability to repair 20 min), requires functional polymerase I activ- these breaks in various bacterial strains was studied ity (20). In contrast, the type III repair process by sedimentation through alkaline sucrose graof the procedure of requires full growth medium conditions at 370C dients by a slight modification McGrath and Williams (15). Cells were labeled with and functional rec gene products (20). 20 yCi of [3H]thymidine per ml (specific activity, 45 ' Present address: Division of Medical Physics, Univer- Ci/mmol; New England Nuclear Corp., Boston, Mass.) for at least three generations, harvested by sity of California, Berkeley, CA 94720. 187
188
ANANTHASWAMY AND EISENSTARK
J. BACTERIOL.
TABLE 1. Strains used Designation
W3110 P3478 JC5029 JC5088 KL168 X9247
Relevant genotypea
polAl
recA56 recB21 recA56 recB21
Other markers
thy thy thi-1 thi-1 thi-1 thi-1
Reference or source
ilv-318 thr-300 rel-1 spc-300 Ailv-318 thr-300 rel-1 spc-300 Xdrm-3 rel-1 Xilv-318 thr-300 spc-300 X-
6 6 5 5 B. J. Bachmann Argonne National
Laboratory W3110 and P3478 are isogenic. JC5029, JC5088, and X9247 are isogenic. KL168 is closely related to JC5029 and JC5088. a
centrifugation, and treated with 0.01 M H202 as described above. After a 10-min treatment at 25°C, 10,ug of catalase per ml (Sigma Chemical Co., St. Louis, Mo.) was added to destroy H202, and a portion was removed quickly and stored in ice at 0°C. The remaining suspension was allowed to stand at room temperature for 15 min, after which time another portion including an untreated control (kept in ice) were gently layered on top of 0.2 ml of lysing solumedium (M9-glucose-Casamino Acids supplemented with the required ingredients) and incubated at 37°C for 40 min. At the end, 0.05-ml samples from each portion including and untreated control (kept in ice) were gently layered on top of 0.2 ml of lysing solution (0.5 M NaOH and 0.05% sodium dodecyl sulfate), which was already layered on a preformed (4.8 ml) 5 to 20% alkaline sucrose gradient (0.5 M NaCl, 0.2 M NaOH and 0.01 M ethylenediaminetetraacetic acid). After standing for 30 min at room temperature for lysis, the gradients were centrifuged at 30,000 rpm for 90 min at 20°C, using an SW50.1 rotor in a Beckman L5-50 ultracentrifuge. Six-drop fractions were collected from the bottom of the tube onto 3MM filter paper disks (Whatman; 2.3 cm) and washed once with 5% trichloroacetic acid and twice with 95% ethanol at room temperature. The dried disks were then placed in vials with about 6 ml of Liquifluor (New England Nuclear Corp.) and counted in a Beckman liquid scintillation counter. The number-average molecular weight (Mn) was calculated, using T7 phage DNA as a marker, according to the formula described by Ley (12).
RESULTS Effect of H202 on survival of bacteria. H202 survival curves of the six strains tested are shown in Fig. 1. The polymerase I-deficient strain P3478 polAl and the X9247 recA recB double mutant were extremely sensitive to H202 compared with the other strains ofE. coli. However, JC5088 recA exhibited increased sensitivity to H202 only in the first log of killing. In contrast, the KL168 recB strain was less sensitive to H202 than the recA strain. Both of the wild-type strains (JC5029 and W3110) exhibited broad shoulders compared with the single-hit kinetics of the mutant strains. An identical control of each strain kept in phosphate buffer alone (no H202) during the entire experiment
z
0
a: c0 0
z
5)
TIME (MINUTES)
FIG. 1. Sensitivity of various E. coli strains to H202. Cells in exponential phase were exposed to 0.01 M H202 in phosphate buffer at room temperature (25°C). JC5029 wild type; W3110 wild type; KL168 recB; JC5088 recA; X9247 recArecB; P3478 polAl.
did not show any loss in colony-forming ability (data not shown). The different sensitivities of the various strains to H202 could be due to the relative differences in their ability to repair the lesions induced by H202. Induction and repair of SS breaks. The existence of two distinct types of repair mechanisms for the repair of SS breaks induced in E. coli after aerobic irradiation with X rays (20), plus our own observation that polAl and rec mutants are considerably more sensitive to H202 than wild-type E. coli, suggested that similar mechanisms might be involved in the repair of
VOL. 130, 1977
REPAIR OF H202-INDUCED BREAKS IN E. COLI DNA
H202-induced SS breaks in the DNA. To demonstrate this, the procedure described by Town et al. (19) was used (see Materials and Methods) to examine polAl-mediated repair (buffer repair or type II repair) and rec-dependent repair (medium-dependent, type Ill repair) processes. Although we are referring to the polAl dependent buffer repair as type II repair and rec-dependent medium repair as type III repair, no distinction has been made to exclude type II and III repairs mediated by polymerase III (9). Recently, Hamelin et al. (9) have shown that DNA polymerase III is involved in type II (in polAl cells) and type III (in polA + and polAl) repairs of gamma-ray-induced DNA SS breaks. The number of SS breaks induced by H202, as well as the number of SS breaks remaining after type II and type III repairs, was calculated from the following equation: number of SS breaks/25 x 108 daltons of DNA -
=25
x
(1
1n
Mnt Mn
where Mnt equals the number-average molecular weight of the treated DNA and Mn0 equals the number-average molecular weight of the untreated control DNA. The induction of SS breaks by H202, followed by the ability to repair these breaks by type II 5.0
W3110
4.0
C'
zA
Z
£
3.0
2.0
1.0
.0 1.0
0.8 FRACTIONAL
0.6
DI STANCE
0.4 0.2 SEDI MENTED
0
FIG. 2. Alkaline sucrose gradient profiles of DNA from E. coli W3110 cells after various treatments. (0) Untreated control (Mn0 = 37 x 10"); (@) 10 min, H202, no incubation (Mnt = 11 x 106); (A) 10 min, H202, 15-min incubation in buffer at 25°C (Mnt = 18 x 106); (A) 10 min, H202, 15-min incubation in buffer at 25°C, 40-min incubation at 370C in full growth medium (Mnt = 30 x 106). The numberaverage molecular weights (Mn) were calculated as described by Ley (12). The direction of sedimentation is from right to left.
189
TABLE 2. Repair of H202-induced SS breaks SS breaks/E. coli genomea Strain
W3110 (wild type) P3478 (polAl) X9247 (recA recB) JC5088 (recA) KL168 (recB) JC5029 (wild type) a
After Aftr AteAr type H0 tyeII and III
treatment II 153 74
239 221 178 148 120
230 182 102 112 51
repairsc
14 154 127 54 37 36
Calculated for each strain from their respective
Mn. and Mn, values mentioned in the legend to Fig. 2 according to the equation given in the text. b In buffer at 25°C for 15 min (requires polA+). c In broth at 37°C for 40 min (requires rec+).
and type III processes in wild-type W3110 and mutant strains, can be inferred from the sedimentation profiles presented in Fig. 2. In addition, calculations of the number of SS breaks per E. coli genome (25 x 108 daltons) (4) after various treatment for each strain (Table 2) clearly indicate the relative differences among various strains in their ability to repair the breaks induced by H202. Brief treatment (10 min) of cells with H202 resulted in the induction of SS breaks to varying degrees, depending on the strain (Table 2). The relative differences in the initial number of observable SS breaks induced by H202 between various strains could be due to one or both of two factors. (i) They could be due to differences in the ability of the various strains to rejoin the SS break rapidly during the 10-min H202 treatment period at 250C. Thus, in repair-proficient strains a certain niumber of SS breaks might have already been repaired by the end of the H202 treatment period. (ii) They could be due to differences in the level of intracellular catalase activity between various strains such that the actual amount of H202 available to react with the DNA in each strain might be different. Although the level of endogenous catalase activity in cell extracts of all the strains has not been measured, at least in two cases (strains JC5088 recA and P3478 polAl) no differences in the level of catalase activity were observed compared with their respective isogenic wild-type parents (JC5029 and W3110) (data not shown). In addition, no significant differences in the level of catalase activity were observed between two wild-type strains (W3110 and JC5029). In this connection, Adler (1) has reported that there is no correlation between the level of catalase activity and sensitivity of E. coli to reagent H202. One of his explanations for the inability of catalase to protect cells
190 ANANTHASWAMY AND EISENSTARK against added H202 is that high concentrations of H202 may inactivate the catalase in cells. Therefore, based on these observations, we can infer that the differences in the initial number of observable SS breaks after peroxide treatment are due to the differences in the ability of the various strains to rejoin the SS breaks during the treatment period. In fact, Town et al. (19) have reported similar differences between strains W3110 pol+ and P3478 polAl in the yield of SS break for a given dose of X rays when irradiated at room temperature but not at OOC. Nevertheless, the relative differences in the ability of the various strains to rejoin the SS breaks induced by H202 become apparent when the number of SS breaks remaining unrepaired after completion of type II and type III repairs is taken into consideration. In the case of P3478 polAl and X9247 recA recB strains, the number of SS breaks remaining unrepaired is significantly higher (154 and 127, respectively) than in the other strains (14 and 54, respectively) (Table 2). In other words, about 60 to 70% of the initial yield of SS breaks is not repaired in these two strains compared with only about 9 to 30% unrepaired breaks in other strains. However, these calculations are only relative and are not strictly accurate because (i) of the differences in the initial yield of SS breaks for reasons discussed above and (ii) the Mn value calculation is extremely sensitive to the small amount of DNA in the upper portions of the gradient (11). DISCUSSION The H202 induction of SS breaks in the DNA (upon treatment of bacteria) supports earlier studies of H202 damage to isolated DNA. The increased sensitivity of polAl, recA recB, and recA strains to H202 compared with the other strains suggests that the lesions induced by H202 are probably not repaired in these mutant strains and that the unrepaired breaks might be the cause for cell death. Similar increased sensitivity of polAl, recA recB, and recA mutants to X rays has been attributed to the inability of these strains to repair the SS breaks induced by X rays (11, 19). Apart from strand breakage, other damages to the DNA such as damages to base and sugar moieties have been thought to be involved in radiation-induced lethality (8, 10, 13, 17, 18). Since H202 is also known to liberate all four bases from the DNA in addition to the breakage of the sugar-phosphate backbone (16), it is quite possible that damages other than strand breakage might also contribute to lethality in bacteria exposed to H202. However, the results of the alkaline sucrose
J. BACTERIOL.
gradient studies (Table 2) reveal that even brief treatment (10 min) of bacteria with low concentrations of H202 (0.01 M) results in the induction of a substantial number of SS breaks. The observed differences in the initial yield of SS breaks by H202 between various strains could be attributed to their relative differences in their ability to rapidly rejoin the breaks during the H202 treatment period. This agrees with the results of Town et al. (19), who reported previously that the differences in the initial yield of SS breaks for a given dose of X rays in W3110 and P3478 polAl strains after aerobic irradiation at room temperature are due to the absence of a rapid repair system in polAl strain, which is present in the wild-type strain. Furthermore, when irradiations were carried out at 0°C, identical yields of SS breaks were obtained in both the strains for a given dose of X rays (19). Analyses of the sucrose gradient sedimentation data indicate that a large number of breaks are left unrepaired after post-treatment incubation in polAl, recA recB, and recA strains compared with the other strains (Table 2). This correlates with the survival data indicating that the increased sensitivities of these mutant strains to H202 are due to their reduced capacity to repair the H202-induced SS breaks. The finding that the recA recB double mutant is comparatively more sensitive to H202 (Fig. 1) and less capable of rejoining the SS breaks than the recA mutant (Table 2) is hard to explain, since the recB mutant exhibits normal capacity to rejoin the H202-induced SS breaks. In contrast, the recA recB double mutant was found to be only slightly more sensitive to X rays than the recA strain, and both strains exhibited more or less identical repair capabilities (11). This discrepancy could be due to differences in the mutant strains used. In summary, we have shown that H202 induces SS breaks in the DNA of bacteria and that these breaks can be repaired by polymerase I-dependent and rec-dependent pathways similar to those already recognized for the repair of X-ray-induced SS breaks in E. coli (20). Furthermore, the results also support the conclusion that the increased sensitivity ofthe mutant strains to H202 is due to their reduced capacity to repair the SS breaks. However, the involvement of polymerases II and III in the repair of H202-induced SS breaks is yet to be ascertained. ACKNOWLEDGMENIS We wish to thank J. Willis for assistance in the calculation of Mn values. This investigation was supported by Public Health Service grant no. 2R01 FD 00 658-05.
VOL. 130, 1977
REPAIR OF H202-INDUCED BREAKS IN E. COLI DNA LITERATURE CITED
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