JouRNAL OF BACTERIOLOGY, Apr. 1979, p. 105-108 0021-9193/79/04-0105/04$02.00/0

Vol. 138, No. 1

Repair of Ultraviolet Light-Damaged Deoxyribonucleic Acid in sbcA Strains of Escherichia coli K-12 DAVID M. SHLAESt AND STEPHEN D. BARBOUR' Department of Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication 28 September 1978

An Escherichia coli strain carrying both rec+ and sbcA has been constructed. Repair of ultraviolet light-induced deoxyribonucleic acid damage was examined by measuring survival and thymine-dimer excision in the rec+ sbcA strain as well as rec+ sbcA+ and recB recC sbcA strains. The sbcA mutation restores normal survival in both recB recC uvrB and recB recC uvrr strains. Excision of thyminecontaining dimers does not occur in uvrB mutants, regardless of the rec or sbcA genotype. Survival, after ultraviolet-light damage, of a rec+ sbcA strain is quantitatively similar to rec+ sbcA+ and recB recC sbcA strains.

Escherichia coli recB and recC mutants form recombinants at a level of about 1% that of rec+ strains (10, 15, 27, 29). Such mutants are moderately sensitive to UV irradiation (27, 29). They are also sensitive to gamma irradiation (16, 29) and to certain DNA-damaging chemicals, including mitomycin C (4,14). recB and recC code for exonuclease V (3, 6, 20, 26). One finds at least two classes of mitomycinresistant mutants of recB+ recC or recB recC+ strains, both of which are recombination proficient and resistant to UV irradiation (4). One class contains true back-mutations in the rec genes, whereas the other class maintains its original rec mutations and has acquired a mutation elsewhere on its genome (4, 19). The latter class can be divided into two subclasses, sbcA and sbcB (4, 18). sbcA mutations are located at min 31 on the E. coli genetic map and are recessive to sbcA+ in merodiploids (2, 19). sbcA strains have acquired a new ATP-independent enzyme, exonuclease VIII (4, 17). The sbcA mutation is thought to lie in a regulatory gene controlling the expression of the structural gene (recE) of exonuclease VIII (19). It has been shown that recombination occurring in sbcA and rec+ strains differs qualitatively. In genetic transformation of E. coli, sbcA recB recC strains yield large numbers of transformants, sbcA rec+ strains yield intermediate numbers, and sbcA+ rec+ strains yield few transformants (10). Also sbcA can efficiently substitute for lambda red to carry out lambda recombination, whereas rec+ cannot (13). We present here studies of repair of UV lightinduced DNA damage in recB recC sbcA, rec+ sbcA, and rec+ sbcA+ strains. t Present addres: Department of Microbiology, Cleveland Metropolitan General Hospital, Cleveland, OH 44109. 105

MATERIALS AND METHODS Bacterial stains. For the bacterial strains used, see Table 1. Media and growth ofbacterial strains. M9 salts, M9 glucose, EM9 glucose, EM9 glycerol, and Luria media have been described elsewhere (7, 8, 29). Plates consisted of liquid medium containing 2% agar. M9 glucose was supplemented with 2 pg of thymidine per ml or 50 ,g of histidine per ml if required. Stock cultures were grown in EM9 glucose from purified single clones to approximately 5 x 108 cells per ml. These stock cultures were stored for a mmum of 1 month at 4°C. To grow an overnight culture, 10 i1 of the stock culture was added to 5 ml of fresh media and placed at 370C with aeration. In the morning the cultures were diluted about 1:75 into fresh media to give an optical density at 650 nm of 0.02. The cultures were then incubated at 370C with aeration. Postirradiation incubations were carried out in EM9 glycerol at 370C with aeration. Determination of exonuclease activity of various strains. The activities of the ATP-dependent and ATP-independent exonucleases were assayed according to Barbour et al.(4). Labeling of cells Cells were grown overnight in EM9 glycerol and diluted into fresh EM9 glycerol in the moming. Cells were labeled for approximately four generations with 20 pCi of methyl-[3H]thymidine (47 Ci/moL Amersham/Searle) in the presence of 250 ,g of deoxyadenosine per ml (5). Excision assay. This method has been described previously (24). Irradiation of cells. This method has been described previously (25). In these experiments the efficiency of dimer formation was 3.5 thymidine dimers per erg per mm2 per chromosome.

RESULTS Activities of exonuclease V and exonuclease VII in various strains. The activities of the ATP-dependent and ATP-independent exonucleases (V and VIII, respectively) as meas-

106

SHLAES AND BARBOUR

J. BACTERIOL.

TABLE 1. E. coli K-12 strains used in these studies Genotype Strain no.

JC4583 JC5176 SDB1305 thySDB1311

Derivation rec

sbc

uvr

gal

his

+ B21 C22 B21 C22 +

+ A6 A6 A6

+ + + +

-

+ +

-

+

See reference See reference Trimethoprim selection JC5176 P1iJC4583 x SDB1305

+ +

Thy+ Exo VIII+ Exo V+a See reference Pl-gal' uvrB5 x JC5176

+

Gal+ UV" Pl gal+ uvrB5 x SDB1311

Reference

(8) (3) This work This work

I

SDB1207 SDB1212

+

B21 C22

+ A6

B5 B5

+ +

(24) This work

I

SDB1211

,+

A6

B5

+

This work

I

Gal+ UV" a Exo, Exonuclease.

ured in crude extracts of various strains are shown in Table 2. These results show that exonuclease V is present when recB+ and recC+ are present and that exonuclease VIII is present when the strain carries the sbcA mutation. The data also show that a rec+ sbcA strain (SDB 1311), constructed by transducing the thy' rec+ alleles into a thy recB recC sbcA recipient, possesses, as expected, both exonuclease V and exonuclease VIII activities. Sensitivity to UV light. We measured survival after exposure to various doses of UV light in uvrB derivatives of strains carrying rec+ and/ or sbcA6 alleles (Fig. 1). Comparing the average shoulders and slopes of recB+ recC+ sbcA6 uvrB5, recB+ recC+ sbcA+ uvrB5, and recB21 recC22 sbcA6 uvrB5, the values from several experiments indicate a difference of less than one target per cell, whereas the slopes differ by about 3 ergs/mm2 per log of killing. The differences shown were reproducible but small. We conclude that (i) recB21 recC22 sbcA6 uvrB5 strains repair UV light-induced damage with an efficiency similar to rec+ uvrB5 strains, and (ii) uvrB5 strains carrying both rec+ and sbcA6 are similar, in efficiency of repair, to uvrB5 strains carrying either recombination function singly. Similar experiments were performed with uvr' derivatives to determine if repair is the same in recB recC sbcA and rec+ sbcA as in rec+ sbcA+ strains (1). Figure 2 indicates that survival among the three uvre derivatives is indistinguishable and therefore that sbcA repair is coordinated with excision as efficiently as rec+ to carry out UV repair synergistically (1). Lack of excision in sbcA uvrB strains. uvrB5 derivatives of sbcA6 recB21 recC22 and sbcA6 rec+ strains were tested for thymine dimer excision. Results (Table 3) indicate that during a 90-min postirradiation incubation no excision

TABLE 2. DNase activity in various strainsa Strain no.

Genotype

-ATP

JC4584 recB recC sbcA+ 0.01 rec+ JC4583 sbcA+ 0.01 recB recC sbcA JC5176 0.06 SDB1311 rec+ sbcA 0.06 a Units of activity expressed per 0.5-mil volume containing 5 ug of protein.

+ATP

0.01 0.15 0.04 0.30 reaction

occurred in either strain. The uvrB5 rec+ sbcA+ strain has previously been shown not to excise (24). Therefore, there is no alternate mechanism in sbcA strains that bypasses the uvrB-controlled step in excision repair.

DISCUSSION In this paper we show that sbcA can substitute for rec+ in repair of UV light-induced damage. Strains carrying both rec+ and sbcA repair similarly to strains carrying either rec+ or sbcA singly. However, differences in recombination as carried out by rec+ and sbcA functions are clear from the work of others. sbcA has been shown to be proficient at recombination during transformation, whereas rec+ is not (10). sbcA is an efficient substitute for the lambda red system of recombination, but rec+ is not (13). Further, recombination and repair can be functionally independent. For example, there is a lack of correlation between recombination and repair in sbcB and xonA mutants of E. coli. sbcB indirectly suppresses recB and recC mutations. Both sbcB and xonA strains lack exonuclease I activity. sbcB recB recC cells are Rec+ UVr, but xonA recB recC cells are Rec- UV' (30). Because qualitative and quantitative differences have been noted for recombination carried out by the sbcA and rec+ recombination functions, a differ-

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REPAIR IN sbcA E. COLI

VOL. 138, 1979

ence in repair capacity might be expected, but was not observed. It seems reasonable to infer that, in recB recC sbcA uvrB and in rec+ sbcA uvrB strains, recombination repair must produce a damage-free product from a damage-containing template without excision taking place. In this regard, repair in these strains must be similar to that in rec+ sbcA+ uvrB strains. Postreplication repair (PRR) is a mechanism that would account for

repair under circumstances where dimners remain distributed in chromosomal DNA (11, 23). PRR occurs in the rec+ sbcA uvrB strain after 200 ergs of UV per mm2 (our unpublished data). It is already known that PRR proceeds at an apparently normal rate in recB recC cells (11, 22, 25). Therefore, if PRR is to account for the UV resistance seen in sbcA revertants and not in recB recC strains, one must postulate a PRR present in recB recC cells which forms an im-

100

recC22

recB21 sbcA uvrB5 \* sbc+ uvrB5 g * rc sbcAe uvrB5 10

C,)

01.0

0

1.0

U/) CO

0

20

40

60

200

0

Dose(e'gS/MM2)

40(

600

Dose(e'gS/MM2)

FIG. 1. Survival after UV irradiation of uvr derivatives of sbcA and sbcA' strains. SDB1207 (0); SDB1212 (A); SDB1211 (A). Points represent average of three experiments.

FIG. 2. Survival after UV irradiation in uvr' derivatives of sbcA and sbcA+ strains. JC4583 (0); JC5176 (A); SDBI311 (U). Points represent average of three experiments.

TABLE 3. Lack of excision in sbcA uvrB strains after 100 ergs of UV radiation per mm' Strain Strain

SDB1212 (recB21 recC22, sbcA6

~~~~irradiatio irra(mition

in thycpm' mine (T)

in dicpm' mers (TT)

5,966,210 2,299.6 4,730,997 1,887.8 4,897,903 1,957.4 SDB1211 (rec+ sbcA6 uvrB5)d 4,792,032 2,089.0 3,723,755 1,327.8 2,944,801 1,770.2 a Cold 5% trichloroacetic acid-precipitable radioactivity. b Ratios in parentheses are corrected for unirradiated control value of 1.2. Refers to corrected values. d Genotype.

uvrB5)d

0 30 90 0 30 90

cpm' in %yb~ ~ 3Hdimers TVxlo, 3.9 (2.7) 4.0 (2.8) 4.0 (2.8) 4.4 (3.2) 3.6 (2.4) 4.2 (3.0)

100 105 105 100 75 94

108

SHLAES AND BARBOUR

perfect product. Such an alternate step has been documented in recB recC cells and is called recF+ (12, 21, 22). sbcA in such a model, by replacing the recB recC product, would supplant the recF+ PRR, with a different step capable of forming a perfect product and acting with an efficiency equal to rec+. Resolution of this must await isolation and characterization of Rec- UV8 mutants of recB recC sbcA strains.

J. BACTERIOL.

14.

15. 16.

ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant GM17329 (awarded to S.D.B.), Public Health Service research career development award GM38140 (awarded to S.D.B.), and Public Health Service training grant GM00171, all three from the National Institute of General Medical Sciences. We thank F. Capaldo, J. Lipson, and G. Ramsey for helpful criticism of parts of this work. We also thank C. Yen for constructing SDB1311 used in this work. LITERATURE CIMD 1. Anderson, J. A., and S. D. Barbour. 1973. Effect of thymine starvation on DNA repair systems of Escherichia coli K-12. J. Bacteriol. 113:114-121. 2. Bachman, B. J., K. B. Low, and A. L, Taylor. 1976. Recalibrated Linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 3. Barbour, S. D., and A. J. Clark. 1970. Biochemical and genetic studies of recombination proficiency in E. coli. I. Enzymatic activity associated with recB+ and recC+ genes. Proc. Natl. Acad. Sci. U.S.A. 65:955-961. 4. Barbour, S. D., H. Nagaishi, A. Templin, and A. J. Clark. 1970. Biochemical and genetic studies of recombination proficiency in E. coli. II. Rec+ revertants caused by indirect suppression of Rec- mutations. Proc. Natl. Acad. Sci. U.S.A. 67:128-135. 5. Boyce, R. P., and R. B. Setlow. 1972. A simple method of increasing the incorporation of thymidine into the DNA of E. coli. Biochim. Biophys. Acta 61:618-620. 6. Buttin, G., and M. Wright. 1968. Enzymatic DNA degradation in E. coli: its relation to synthetic processes at the chromosomal level. Cold Spring Harbor Symp. Quant. Biol. 33:259-269. 7. Capaldo, F. N., and S. D. Barbour. 1975. DNA content, synthesis and integrity in dividing and non-dividing cells in Rec- strains of Escherichia coli K12. J. Mol. Biol. 91:53-66. 8. Capaldo-Kimball, F., and S. D. Barbour. 1971. Involvement of recombination genes in growth and viability of Escherichia coli K-12. J. Bacteriol. 106:204-212. 9. Clark, A. J. 1967. The beginning of a genetic analysis of recombination proficiency. J. Cell Physiol. 70:165-180. 10. Cosloy, S. D., and M. Oishi. 1973. The nature of the transformation process in Escherichia coli K12. Mol. Gen. Genet. 24:1-10. 11. Ganesan, A. K. 1974. Persistance of pyrimidine dimers during post-replication repair in ultraviolet light irradiated E. coli K12. J. Mol. Biol. 87:103-119. 12. Ganesan, A. K., and P. C. Seawell. 1975. The effect of lexA and recF mutations on post-replication repair and DNA synthesis in E. coli K12. Mol. Gen. Genet 141: 189-205. 13. Gottesman, M. M., M. E. Gottesman, S. Gottesman,

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and M. Geliert. 1974. Characterization of bacteriophage A reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions. J. Mol. Biol. 88:471-487. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. Radiat. Res. Suppl. 6:156-184. Howard-Flanders, P., and L. Theriot. 1966. Mutants of E. coli K12 defective in DNA repair and in genetic recombination. Genetics 53:1137-1150. Kapp, D. S., and K. C. Smith. 1970. Repair of radiationinduced damage in Escherichia coli. II. Effect of rec and uvr mutations on radiosensitivity and repair of Xray-induced single-strain breaks in deoxyribonucleic acid. J. Bacteriol. 103:49-54. Kushner, S. R., IL Nagaishi, and A. J. Clark. 1974. Isolation of exonuclase VIII: the enzyme associated with the sbcA indirect suppressor. Proc. Natl. Acad. Sci. U.S.A. 71:3593-3597. Kushner, S. R., H. Nagaishi, A. Templin, and A. J. Clark. 1971. Genetic recombination in E. coli: the role of exonuclease I. Proc. Natl. Acad. Sci. U.S.A. 68:824827. Uoyd, R. G., and S. D. Barbour. 1974. The genetic location of the sbcA gene of Escherichia coli. Mol. Gen. Genet. 134:157-171. Oishi, M. 1969. An ATP-dependent DNAse from E. coli with a possible role in genetic recombination. Proc. Natl. Acad. Sci. U.S.A. 64:1292-1299. Rothman, R. H., and A. J. Clark. 1977. The dependence of post replication repair on uvrB in a recF mutant of E. coli K12. Mol. Gen. Genet. 155:279-286. Rothman, R. H., T. Kato, and A. J. Clark. 1975. The beginning of an investigation of the role of recF in the pathways of metabolism of U.V. irradiated DNA in E. coli, p. 2&3-291. In P. C. Hanawalt and R. B. Setlow (ed.), Molecular mechanisms for repair ofDNA. Plenum Publishing Corp., New York. Rupp, W. D., and P. Howard-Flanders. 1968. Discontinuities in the DNA synthesized in an excision defective strain of E. coli following ultraviolet irradiation. J. Mol. Biol. 31:291-303. Shlaes, D. M., J. A. Anderson, and S. D. Barbour. 1972. Excision repair properties of isogenic rec mutants of Escherichia coli K-12. J. Bacteriol. 111:723-730. Smith, K. C., and D. H. C. Muen. 1970. Repair of radiation induced damage in E. coli. I. Effect of rec mutations in post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51:459-472. Tomizawa, J.-I., and H. Ogawa. 1972. Structural genes of ATP-dependent DNAse of E. coli. Nature (London) New Biol. 239:14-16. Willets, N. S., and A. J. Clark. 1969. Characteristics of some multiply recombination-deficient strains of E8cherichia coli. J. Bacteriol. 100:231-239. Willets, N. S., A. J. Clark, and B. Low. 1969. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J. Bacteriol. 97:244-249. Willets, N. S., and D. W. Mount. 1969. Genetic analysis of recombination-deficient mutants of Escherichia coli K-12 carrying rec mutations cotransducible with thyA. J. Bacteriol. 100:923-934. Yajko, D. M., M. C. Valentine, and B. Weiss. 1974. Mutants of E. coli with altered DNAses. II. Isolation and characterization of mutants for exonuclease I. J. Mol. Biol. 85:323-343.

Repair of ultraviolet light-damaged deoxyribonucleic acid in sbc-A strains of Escherichia coli K-12.

JouRNAL OF BACTERIOLOGY, Apr. 1979, p. 105-108 0021-9193/79/04-0105/04$02.00/0 Vol. 138, No. 1 Repair of Ultraviolet Light-Damaged Deoxyribonucleic...
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