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Uvr-independent repair of 8-methoxypsoralen crosslinks in Escherichia coli: Evidence for a recombinational process
Mutation Research, 146 (1985) 135-141 DNA Repair Reports
135
Elsevier MTR06105
Uvr-independent repair of 8-methoxypsoralen crosslinks in Escherichia coli: Evidence for a recombinational process Marinus Cupido and Bryn A. Bridges * MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex BN1 9RR (Great Britain) (Received 4 December 1984) (Revision received 8 February 1985) (Accepted 19 February 1985)
Summary On the basis of survival data, repair of 8-methoxypsoralen DNA crosslinks in Escherichia coli strains lacking a functional uvrABC endonuclease, is shown to require the products of the recA, recB, recF and recN genes. Bacteria, grown under conditions where most cells contain only a single genome, show no evidence of crosslink repair. Similarly, bacteriophage ~ shows evidence of crosslink repair only in SOS-induced cells, and only at multiplicities of infection greater than 1. The requirement for rec + genes may be partly ascribed to the need for a functional SOS response, but taken together, the results suggest a recombinational step involving a homologous region of DNA may occur during uvr-independent crosslink repair.
8-Methoxypsoralen (8-MOP) complexes readily with DNA in the dark by intercalation between adjacent base pairs. When this complex is irradiated with 340-360 nm light (UVA) the 3,4-carbon or the 4',5'-carbon double bonds in the psoralen may form a cyclic adduct with the 5',6'-carbon double bond of a pyrimidine in the DNA. A proportion of the 4',5' adducts may use another photon to react with the 3,4-carbon double bond of the psoralen and connect it with a pyrimidine in the opposite strand to form a DNA interstrand crosslink (Dall'Acqua, 1977). Psoralen monoadducts are bypassed during DNA replication in E. coli (Piette and Hearst, 1983), S. cerevisiae (Chanet et al., 1983) and in cultured human lymphocytes (Cohen et al., 1981) but interstrand crosslinks provide a block to DNA replication in all cases. * Tho whom correspondence should be addressed.
Repair of crosslinks in E. coli has been extensively studied by Cole and colleagues (Cole, 1970, 1973; Cole and Zusman, 1970; Cole et al., 1976; Sinden and Cole, 1978). Based on in vitro and in vivo studies they proposed the following model for repair of crosslinks. The uorABC endonuclease of E. coli cuts one DNA strand on the 3' side of a crosslink followed by a cut on the 5' side of the crosslink made by the 5'-3' exonuclease activity of DNA polymerase I assisted by the uvrD gene product (DNA helicase II, Kumura et al., 1983). (It was established later (Sancar and Rupp, 1983) that the uvrABC endonuclease is able to make two cuts by itself when the damage consists of UV-induced photoproducts.) The single-stranded gap thus generated, is enlarged to about 1000 nucleotides in size and subsequently filled via homologous recombination (Cole et al., 1978). It was shown in our laboratory that crosslinks are repaired, albeit at much reduced efficiency, in
0167o8817/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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bacteria that lack the uvrABC endonuclease (Bridges and Von Wright, 1981). Like crosslink repair in uvrABC-proficient bacteria, this repair is dependent on the function of the recA gene. It is also dependent on the rep gene function and it is inhibited by acriflavine and the muc + genes on plasmid pKM101 (Bridges, 1984; Cupido and Bridges, 1985). A model was proposed for removal of crosslinks and monoadducts in the replication fork by an N-glycosylase. The resulting apyrimidinic site could be dealt with during DNA replication in a process involving either recombination or bypass under the influence of the u m u C D gene products (Bridges and Von Wright, 1981; Bridges, 1984). Repair of DNA crosslinks appears to be inducible and may well be part of the general response of E. coli to the presence of damaged DNA, usually referred to as the SOS response (Radman, 1975; Witkin, 1976). The SOS response is regulated by two proteins: the lexA gene product that represses the genes involved in the SOS response
and the recA gene product that is activated to a protease as DNA replication is obstructed (Roberts and Devoret, 1983) and which then inactivates the lexA protein (reviewed by Little and Mount, 1982). Sinden and Cole (1978) showed that there is no crosslink repair in a lexA (Ind-) strain, in which the SOS response cannot be induced, and Cupido and Bridges (1985) showed that in a recA441 (formerly tif-1) uvrA double mutant crosslink repair was more efficient at 43°C where the SOS response is fully induced than at 30°C where the SOS response is still dependent on an inducing signal. In the present paper, we have further characterized repair of psoralen-plus-UVA (PUVA)-induced crosslinks in excision-deficient strains of both the E. coli K12 and E. coli B / r families. The results show a requirement for 4 recombination genes and suggest that recombination with a second chromosome may be involved in repair of DNA interstrand crosslinks, even when repair is not initiated by the uvrABC endonuclease.
TABLE 1
Escherichia coli STRAINS Strain
Genotype
(A) Escherichia eoli B / r derivatives WP2 uvrA uvrA155 trpE65 lon-l l sulA32 CM 1131 as WP2 uvrA but tna- 300: : TnlO revF143 CMl145 WP100
as WP2 uvrA but tyrAl6::TnlO recN262 as WP2 uvrA but recA1 uvrA155 trpE65
(B) Escherichia coli K12 derivatives AB1886 uvrA6 thr- 1 leu- 6 proA6 his- 4 thi- 1 argE3 galK2 ara- 14 xyl-5 mtl tsx33 strA31 supE44 AB2480 as AB1886 but recA 13 AB3072 as AB1886 but recB21 SP263 as AB1886 but tyrA16: :TnlO SP264 asAB1886 but tyrA16: :TnlO recN262 TK907 as AB1886 but recA - 441 sfiA- 11 proA-2 ilv-325 argE + CMll50 as AB1886 but tna- 300::TnlO recF143 CMll37 as AB1886 but proA2 ilv- 325 argE + srl 300: : Tnl O RecA56 All transductions were carried out with P1 cml, clrlO0 (Miller, 1972).
Source or derivation Hill, 1965 P] JC 1 2 3 3 4 x W P 2 uvrA (Southworth and Bridges, 1984) P1 S P 2 6 4 × W P 2 uvrA E.M. Witkin
Howard-Flanders and Theriot, 1966
P1 AB2470 x AB1886 Picksley et al., 1984 Picksley et al., 1984 T. Kato P1 JC12334 x AB1886 P1 JC10240 x TK603
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Materials and methods
Micro-organisms The strains of Escherichia coli used are listed in Table 1. Media Bacteria were grown with aeration to about 2 x 108/ml in Oxoid nutrient broth No. 2 and plated on the same medium solidified with 1% agar. In some experiments bacteria were grown in the minimal medium of Davis and Mingioli (1950) supplemented with (per litre) 50 mg thymine and either 10 mg tryptophan ( B / r strains) or with 100 mg threonine, leucine, proline, arginine and histidine and 0.25 mg thiamine (K12 strains). As stated in the text, in some experiments the 0.4% glucose was replaced with 0.4% or 0.04% aspartic acid, neutralized with NaOH. Where not specifically stated, incubation temperature was 37°C. Irradiation and experimental procedure Bacteria were suspended in phage buffer (Boyle and Symonds, 1969) at room temperature at a density of around 2 x 108/ml. 8-Methoxypsoralen (Sigma) was added to a final concentration of 20 t t g / m l and the suspension allowed to stand for 5 rain in the dark. Samples were irradiated with UVA 10.5 cm from a Philips 20 W "Blacklight" lamp at a fluence of 2.3 X 10 -4 w / c m 2. After initial exposure a 10 000-fold dilution was made in phage buffer and a further 40-rain exposure to UVA was given. All experiments were carried out at least 3 times with consistent results. Although there appeared to be a considerable day-to-day variation, the ratio in sensitivity between relevant strains remained constant in different experiments.
counted in a liquid scintillation counter. The bacteria were sonicated for 2 × 15 sec and spun at 1 2 0 0 0 X g for 15 rain. The supernatant was counted again in the scintillation counter to determine the recovery of the DNA (usually between 40% and 70%). The DNA content in the samples was determined with the Hoechst Dye procedure (Labarca and Paigen, 1980). DNA content per cell was calculated by dividing the DNA content over the number of initial cells adjusting for the loss made during sedimentation of cell debris after the sonication. Results
Repair in recombination-deficient strains We have tested the effect on crosslink repair of a number of alleles that reduce the ability of the bacteria to perform recombination. When introduced into strain AB1886 the alleles recA56, recB21, recF143 and recN262 all resulted in reduced survival after the induction of crosslinks. The results in strain WP2 uvrA are similar except for the fact that the recB21 allele was not tested in
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Estimation of DNA content per cell Bacteria were resuspended in T r i s - E D T A buffer (50 mM Tris, 2 mM EDTA, pH 7.6) and from each culture samples were counted in a bacterial counting chamber. Density was usually around 2 X 108/ml. All further manipulations were done at 0°C. An equal volume of the same medium containing 800 # g / m l freshly dissolved egg-white lysozyme (Sigma) was added and incubated for 10 min to form spheroplasts. Ceils labelled with [3H]thymidine were added and a sample was
Fig. 1. Survival of recombination-deficient derivatives of
Escherichia coli K12 (A) or B / r (B) after the induction of 8-MOP crosslinks (representative experiments). (A) I,, AB1886; O, CM1137 (recA56); D, AB3072 (recB21l); I, CMl150 (reeF143); A, SP264 (recN262). (B) e, WP2 uvrA; O, WP100 (fecAl); I , CMl131 (reeF143); A, CMl145 (recN262). The abscissa represents seconds of exposure to UVA at a fluence of 2.3 x 10 -4 W / m 2 in the presence of 20 /~g/ml 8-MOP. After exposure to UVA 8-MOP was diluted out at least 10 -4 and the samples were exposed to UVA for a further 40 rain to convert as many psoralen adducts as possible to crosslinks. It is assumed that the yield of crosslinks is proportional to the initial UV exposure.
138
this background (Fig. 1). Crosslink repair may be an inducible function and is probably a part of the more general SOS response. It was reported that the recF mutation decreases induction of SOS by ultraviolet light and possibly crosslinks, but not with nalidixic acid (McPartland et al., 1980). Therefore, we have tried to assess whether the sensitivity to crosslinks of CMl131, a derivative of WP2 containing uvrA6 recF143, was the result of a poor induction of the SOS response by pretreating the cells with 50 ~ g / m l nalidixic acid for 60 min at 37°C prior to the induction of crosslinks. It is shown in Fig. 2 that this treatment partly restored the capacity of recF-deficient cells to repair crosslinks, although repair in WP2 uvrA recF + also was stimulated. This suggests that the low number of crosslinks induced in these experiments is in itself not a strong inducer of the SOS response even in rec + cells.
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Fig. 2.Survival of the E. coli B / r strain WP2 uvrA (circles) and reeF143 derivative CMl131 (squares) after induction of crosslinks. The bacteria were either grown in nutrient broth (solid symbols) or treated with 5 0 / ~ g / m l nalidixic acid for 1 h at 37°C (open symbols) before crosslinks were induced as described in the legend to Fig. 1. a
Possible requirement for multiple genomes The requirement for several recombination genes suggested the possible involvement of a recombination event, as is believed to occur during crosslink repair in excision-proficient bacteria. We therefore sought to investigate whether there is a requirement for a second, homologous genome. In the first set of experiments, bacteria were grown for several generations in medium where the main carbon source was aspartic acid neutralized with N a O H , instead of glucose. It has been reported that 0.4% aspartic acid resulted in the presence of only one or slightly more than one genome per cell, conditions in which recombination with another homologous D N A molecule cannot occur (Krasin and Hutchinson, 1977). We could not reproduce this exactly, but reduction of the aspartate concentration in the medium from 0.4% to 0.04% resulted in an increase of the generation time of the bacteria from 1 to 4 h. The average D N A content was reduced from 43.0 _+ 9.5 femto (10 15) g r a m / b a c t e r i u m to 5.6 ± 3.2 fg/bacterium in E. coli WP2 uurA and from 10.4 _+ 2.0 f g / b a c t e r i u m to 5.0 ± 0.9 fg/bacterium in E. coli AB1886. Assuming that the E. coli genome equals 4.6 fg of D N A (Bridges, 1971) this corresponds with a reduction from 9.3 _+ 2.1 to 1.2 ± 0.7 geh o m e s / b a c t e r i u m in WP2 uorA and from 2.3 _+ 0.4 to 1.1 +_ 0.2 genomes/bacterium in AB1886. When the D N A in cells grown on 0.04% aspartic acid was crosslinked with 8-MOP and UVA a higher mortality was observed than in cells grown on glucose or on 0.4% aspartic acid (Fig. 3). This is consistent with a requirement for a second genome, although other trivial explanations cannot be excluded. It may be noted that the hypersensitivity of cells grown on aspartate is in contrast to the general trend that (multigenomic) cells are more resistant to crosslinks in minimal growth medium (Bridges, 1984). The resistant tail in the line representing AB1886 cells grown on 0.04% aspartate medium may reflect a fraction of the cells containing more than one genome. Another approach was to study the survival of crosslinked phage ~ after infection of a uorA-deficient host. We did not find any evidence for crosslink repair when the survival curves were made with a multiplicity of infection (m.o.i.) lower than 1, confirming the results of Bridges and Von
139 ,
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Fig. 3. Survival of E. coli AB1886 (A) and WP2 uorA (B), after induction of 8-MOP crosslinks (representative experiment), e, bacteria grown in minimal medium with 0.4% glucose; t2, bacteria grown in minimal medium with 0.4% aspartic acid; ©, bacteria grown in minimal medium with 0.04% aspartic acid (resulting in around 1 genome/bacterium). Irradiation procedures and UVA doses are the same as in Fig. 1.
05
Wright (1981). When experiments were done with a m.o.i, of 2 - 4 there was a small but significant increase in survival of incoming ~ phage if the cells contained the recA441 mutation and were grown at 42°C (Fig. 4). When the recA441 host strain was growth at 30°C, the survival of crosslinked ~, phage was identical to the survival in the experiments performed at low m.o.i. The experiments were performed in a strain with the recA441 (formerly tif-1) mutation, as it was reasoned that crosslink repair might be a response induced by the presence of damaged D N A in the bacteria. This could lead to more efficient repair in the bacteria that received a stronger inducing signal, i.e the cells infected with high m.o.i., but in a recA441 mutant the SOS response is induced by a temperature shift from 30°C to 42°C. Moreover, one can assume that the m.o.i, is the same at both temperatures and no corrections have to be made to compensate for the increased possibility of the bacteria to be infected by undamaged particles at high m.o.i. (Hall, 1982). These experiments are consistent with the hypothesis that the presence of at least two genomes in uvrA-deficient bacteria allows more repair of D N A crosslinks.
Discussion Z O V-t.D < r~ LL tD
zo-1 >
0.OS
0.01t
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150
I
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300 1,50 INITIAL UVA DOSE
I
600
Fig. 4. Survival of phage 2~ after induction of crosslinks. Phage particles at a density of 109/ml were exposed to UVA in the presence of 20 /~g/ml 8-MOP. After dilution of the 8-MOP and conversion of monoadducts to crosslinks as mentioned in the legend to Fig. 1, the phage were absorbed to E. coli TKg07 (recA441, uorA6) and then plated on a lawn of E. coli AB2480 (recA13, uorA6) to prevent plaque formation by unabsorbed phage. Incubation temperatures were either 30°C (©) or 42°C
(o).,
Cole et al. (1978) have shown that a recA-deficient but excision-proficient strain survives only 0.5-1.6 crosslinks/genome, whereas recB or recF mutants survive between 5.5 and 13 crosslinks/genome. We have found in an exision-deficient background, that recA, recB and recF mutants do not differ significantly from each other in sensitivity to crosslinks. All are more or less equally hypersensitive to crosslinks. It appears that crosslink repair, initiated by the uvrABC endonuclease can be completed via recombination in the absence of either the recB or the recF gene product, whereas in an excision-deficient strain, both these gene products are required for repair. In these strains, the initial step in crosslink repair might be the induction of a single-strand break close to the crosslink by another repair enzyme such as endonuclease III (Radman, 1976; Gates and Linn, 1977) or the recF-dependent protein Z endonuclease (Krivonogov, 1984), followed by a second break in the opposite strand. This may possibly explain the
140
requirement for the recN gene product, which is shown to be associated with repair of DNA double-strand breaks (Lloyd et al., 1983; Picksley et al., 1984). The recB gene product has also been reported to be required for repair of double-strand breaks (Wang and Smith, 1983). It is also possible, notwithstanding, to avoid a double-strand break if single incisions are made in each strand on opposite sides of the crosslink. The free ends thus produced could conceivably initiate crossovers with a homologous duplex on either side of the crosslink. Although there is no evidence on this point, the initial steps of this repair may be the same as those in the repair of psoralen monoadducts, shown to be present in uc'rA-deficient bacteria (Bridges and Stannard, 1982; Bridges, 1983). The role of the recF gene product may be an indirect one, as it is supposed to affect the "specificity of the recA protein binding to different effectors" (Karu and Belk, 1982). A number of observations support this hypothesis. (1) Several mutants that suppress the recF143 phenotype have been mapped in the recA gene (Volkert and Hartke, 1984), (2) the recA441 (formerly tif-1) mutation also suppresses the recF143 defect (Thomas and Lloyd, 1983, Volkert et al., 1984) and concomitantly makes recA protein more efficient in interacting with DNA and nucleoside triphosphates (Phizicky and Roberts, 1981), (3) the induction of the SOS response with nalidixic acid partly restores the capacity of a recF143 uvrA6 strain to repair crosslinks. The fact that recA, recB, recF and recN mutations all block repair of 8-MOP crosslinks in uvrA-deficient bacteria suggests that a recombination step might be involved. Other experiments have produced data consistent with the need for a second, homologous genome in order to carry out such repair. Taken together, these two lines of evidence support a recombinational model, but biochemical evidence will be needed before this can be regarded as established. We are clearly still some way from a functional understanding of crosslink repair in excision-deficient bacteria.
Acknowledgement Marinus Cupido is a recipient of a European Community Fellowship.
References Boyle, J.M., and N. Symonds (1969) Radiation sensitive mutants of T4D. I., T4y: a new radiation sensitive mutant: effect of the mutation on radiation survival, growth and recombination, Mutation Res., 8, 431 439. Bridges, B.A. (1971) RecA+-dependent repair of gamma-ray damage to Escherichia coli does not require recombination between existing homologous chromosomes, J. Bacteriol., 108, 944-945. Bridges, B.A. (1983) Psoralens and serendipity: Aspects of the genetic toxicology of 8-methoxypsoralen, Environ. Mutagen., 5, 329-339. Bridges, B.A. (1984) Further characterization of repair of 8methoxypsoralen crosstinks in UV-excision-defective Escherichia coli, Mutation Res., 132, 153 160. Bridges, B.A., and M. Stannard (1982) A new pathway for repair of cross-linkable 8-methoxypsoralen mono-adducts in uvr strains of Escherichia coli, Mutation Res., 92, 9 14. Bridges, B.A., and A.J. yon Wright (1981) Influence of mutations at the rep gene on survival of Escherichia coli following ultraviolet light irradiation or 8-methoxypsoralen photosensitization, Evidence for a recA + rep+-dependent pathway for repair of DNA crosslinks, Mutation Res., 82, 229-238. Chanet, R., C. Cassier, N. Magaha-Schwenke and E. Moustacchi (1983) Fate of photo-induced 8-methoxypsoralen monoadducts in yeast. Evidence for bypass of these lesions in the absence of excision repair, Mutation Res., 112, 201 214. Cohen, L.F., K.H. Kraemer, H.L. Waters, K.W. Kohn and D.L. Glaubiger (1981) DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation, Mutation Res., 80, 347-356. Cole, R.S. (1970) Light-induced cross-linking of DNA in the presence of a furocoumarin (psoralen); studies with phage lambda, Escherichia coli, and mouse leukemia cells, Biochim. Biophys. Acta, 217, 30-39. Cole, R.S. (1973) Repair of DNA containing interstrand crosslinks in Escherichia coli, Sequential excision and recombination, Proc. Natl. Acad. Sci. (U..S.A.), 70, 1064-1068. Cole, R.S., and D. Zusman (1970) Sedimentation properties of phage DNA molecules containing light-induced psoralen crosslinks, Biochim. Biophys. Acta, 224, 660-662. Cole, R.S., D, Levitan and R.R. Sinden (1976) Removal of psoralen interstrand cross-links from DNA of Escherichia coli, Mechanism and genetic control, J. Mol. Biol., 103, 39-59. Cole, R.S., R.R. Sinden, G.H. Yoakum and S. Broyles (1978) On the mechanism for repair of cross-linked DNA in E. coli treated with psoralen and light, in: P.C. Hanawalt, E.C. Friedberg and C.F. Fox (Eds.), ICN-UCLA Symposia on Molecular and Cellular Biology, DNA Repair Mechanisms, Vol. 9, Academic Press, New York, pp. 287-290. Cupido, M., and B.A. Bridges (1985) Paradoxical behaviour of pKM101; inhibition of uor-independent crosslink repair in Escherichia coli by rnuc gene products, Mutation Res., 145, 49-53.
141 Dall'Acqua, F. (1977) New chemical aspects of the photoreaction between psoralen and DNA, in: A. Castellani (Ed.), Research in Photobiology, Plenum, New York, pp. 245-255. Dall'Acqua, F., S. Marciani and G. Rodrighiero (1970) Interstrand cross-linkages occurring in the photoreaction between psoralens and D N A , FEBS Lett., 9, 121-123. Davis, B.D., and E.S. Mingioli (1950) Mutants of Escherichia coli requiring methionine or vitamin B12, J. Bacteriol., 60, 17-28. Gates, F.T., and S. Linn (1977) Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, nitrous acid, or X-rays, J. Biol. Chem., 252, 2802-2807. Hall,. J.D. (1982) Repair of psoralen-induced crosslinks in cells multiply infected with SV40, Mol. Gen. Genet., 188, 135-138. Hill, R.F. (1965) Ultraviolet-induced lethality and reversion to prototrophy in Escherichia coli strains with normal and reduced dark repair ability, Photochem. Photobiol., 4, 563-568. Howard-Flanders, P., and L. Theriot (1966) Mutants of Escherichia coli K12 defective in DNA repair and in genetic recombination, Genetics, 53, 1137-1150. Karu, A.E., and E.D. Belk (1982) Induction of E. coli recA protein via recBC and alternate pathways: Quantitation by enzyme-linked immunoabsorbent assay (ELISA), Mol. Gen. Genet., 185, 275-282. Krasin, F., and F. Hutchinson (1977) Repair of DNA doublestrand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome, J. Mol. Biol., 116, 81-98. Krivonogov, S.V. (1984) The recF-dependent endonuclease from Escherichia coli K12, Formation and resolution of pBR322 DNA multimers, Mol. Gen. Genet., 196, 105-109. Kuhn, B., M~ Abdel-Monem and H. Hoffmann-Berling (1979) DNA helicases, Cold Spring Harbor Symp. Quant. Biol., 43, 63-67. Kumura, K., K. Oeda, M. Akiyama, T. Horiuchi and M. Sekiguchi (1983) The uvrD gene of E. coli: Molecular cloning and expression, in: E.C. Friedberg and B.A. Bridges (Eds.), UCLA Symposia on Molecular and Cellular Biology, Cellular Responses to DNA Damage, Vol. 11, Liss, New York, pp. 51-62. Labarca, C., and K. Paigen (1980) A simple, rapid and sensitive DNA assay procedure, Anal. Biochem., 102, 344-352. Little, J.W., and D. Mount (1982) The SOS regulatory system of Escherichia coli, Cell, 29, 11-22. Lloyd, R.G., S.M. Picksley and C. Prescott (1983) Inducible expression of a gene specific for the recF pathway for recombination in Escherichia coli K12, Mol. Gen. Genet., 190, 162-167. McPartland, A., L. Green and H. Echols (1980) Control of recA gene RNA in E. coli: Regulatory and signal genes, Cell, 20, 731-737.
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, p. 203. Phizicky, E.M., and J.W. Roberts (1981) Induction of SOS functions: regulation of proteolytic activity of E. coli Rec protein by interaction with D N A and nucleoside triphosphate, Cell, 25, 259-267. Picksley, S.M., P.V. Attfield and R.G. Lloyd (1984) Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product, Mol. Gen. Genet., 195, 267-274. Piette, J.G., and J.E. Hearst (1983) Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5540-5544. Radman, M. (1975) SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, in: P. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, part A, Plenum, New York, pp. 355-367. Radman, M. (1976) An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA, J. Biol. Chem., 251, 1438-1445. Roberts, J., and R. Devoret (1983) Lysogenic induction, in: Lambda 2, Cold Spring Harbor Laboratory, New York, pp. 123-144. Sancar, A., and W.D. Rupp (1983) A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33, 249-260. Sinden, R.R., and R.S. Cole (1978) Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: Effects of mutations influencing genetic recombination and DNA metabolism, J. Bacteriol., 136, 538-547. Southworth, M.W., and B.A. Bridges (1984) Influence of reeF on spontaneous mutation in Escherichia coli, Mutation Res., 140, 67-69. Thomas, A., and R.G. Lloyd (1983) Control of recA dependent activities in Escherichia coli: a possible role for the recF product, J. Gen. Microbiol., 129, 681-686. Volkert, M.R., and M.A. Hartke (1984) Suppression of Escherichia coli recF mutations by recA-linked srfA mutations, J. Bacteriol., 156, 1093-1098. Volkert, M.R., L.J. Margossian anbd A.J. Clark (1984) Two component suppression of recF143 by recA441 in Escherichia coli K12, J. Bacteriol., 160, 702-705. Wang, T.V., and K.C. Smith (1983) Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB, J. Bacteriol., 156, 1093-1098. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.
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Elsevier MTR06105
Uvr-independent repair of 8-methoxypsoralen crosslinks in Escherichia coli: Evidence for a recombinational process Marinus Cupido and Bryn A. Bridges * MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex BN1 9RR (Great Britain) (Received 4 December 1984) (Revision received 8 February 1985) (Accepted 19 February 1985)
Summary On the basis of survival data, repair of 8-methoxypsoralen DNA crosslinks in Escherichia coli strains lacking a functional uvrABC endonuclease, is shown to require the products of the recA, recB, recF and recN genes. Bacteria, grown under conditions where most cells contain only a single genome, show no evidence of crosslink repair. Similarly, bacteriophage ~ shows evidence of crosslink repair only in SOS-induced cells, and only at multiplicities of infection greater than 1. The requirement for rec + genes may be partly ascribed to the need for a functional SOS response, but taken together, the results suggest a recombinational step involving a homologous region of DNA may occur during uvr-independent crosslink repair.
8-Methoxypsoralen (8-MOP) complexes readily with DNA in the dark by intercalation between adjacent base pairs. When this complex is irradiated with 340-360 nm light (UVA) the 3,4-carbon or the 4',5'-carbon double bonds in the psoralen may form a cyclic adduct with the 5',6'-carbon double bond of a pyrimidine in the DNA. A proportion of the 4',5' adducts may use another photon to react with the 3,4-carbon double bond of the psoralen and connect it with a pyrimidine in the opposite strand to form a DNA interstrand crosslink (Dall'Acqua, 1977). Psoralen monoadducts are bypassed during DNA replication in E. coli (Piette and Hearst, 1983), S. cerevisiae (Chanet et al., 1983) and in cultured human lymphocytes (Cohen et al., 1981) but interstrand crosslinks provide a block to DNA replication in all cases. * Tho whom correspondence should be addressed.
Repair of crosslinks in E. coli has been extensively studied by Cole and colleagues (Cole, 1970, 1973; Cole and Zusman, 1970; Cole et al., 1976; Sinden and Cole, 1978). Based on in vitro and in vivo studies they proposed the following model for repair of crosslinks. The uorABC endonuclease of E. coli cuts one DNA strand on the 3' side of a crosslink followed by a cut on the 5' side of the crosslink made by the 5'-3' exonuclease activity of DNA polymerase I assisted by the uvrD gene product (DNA helicase II, Kumura et al., 1983). (It was established later (Sancar and Rupp, 1983) that the uvrABC endonuclease is able to make two cuts by itself when the damage consists of UV-induced photoproducts.) The single-stranded gap thus generated, is enlarged to about 1000 nucleotides in size and subsequently filled via homologous recombination (Cole et al., 1978). It was shown in our laboratory that crosslinks are repaired, albeit at much reduced efficiency, in
0167o8817/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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bacteria that lack the uvrABC endonuclease (Bridges and Von Wright, 1981). Like crosslink repair in uvrABC-proficient bacteria, this repair is dependent on the function of the recA gene. It is also dependent on the rep gene function and it is inhibited by acriflavine and the muc + genes on plasmid pKM101 (Bridges, 1984; Cupido and Bridges, 1985). A model was proposed for removal of crosslinks and monoadducts in the replication fork by an N-glycosylase. The resulting apyrimidinic site could be dealt with during DNA replication in a process involving either recombination or bypass under the influence of the u m u C D gene products (Bridges and Von Wright, 1981; Bridges, 1984). Repair of DNA crosslinks appears to be inducible and may well be part of the general response of E. coli to the presence of damaged DNA, usually referred to as the SOS response (Radman, 1975; Witkin, 1976). The SOS response is regulated by two proteins: the lexA gene product that represses the genes involved in the SOS response
and the recA gene product that is activated to a protease as DNA replication is obstructed (Roberts and Devoret, 1983) and which then inactivates the lexA protein (reviewed by Little and Mount, 1982). Sinden and Cole (1978) showed that there is no crosslink repair in a lexA (Ind-) strain, in which the SOS response cannot be induced, and Cupido and Bridges (1985) showed that in a recA441 (formerly tif-1) uvrA double mutant crosslink repair was more efficient at 43°C where the SOS response is fully induced than at 30°C where the SOS response is still dependent on an inducing signal. In the present paper, we have further characterized repair of psoralen-plus-UVA (PUVA)-induced crosslinks in excision-deficient strains of both the E. coli K12 and E. coli B / r families. The results show a requirement for 4 recombination genes and suggest that recombination with a second chromosome may be involved in repair of DNA interstrand crosslinks, even when repair is not initiated by the uvrABC endonuclease.
TABLE 1
Escherichia coli STRAINS Strain
Genotype
(A) Escherichia eoli B / r derivatives WP2 uvrA uvrA155 trpE65 lon-l l sulA32 CM 1131 as WP2 uvrA but tna- 300: : TnlO revF143 CMl145 WP100
as WP2 uvrA but tyrAl6::TnlO recN262 as WP2 uvrA but recA1 uvrA155 trpE65
(B) Escherichia coli K12 derivatives AB1886 uvrA6 thr- 1 leu- 6 proA6 his- 4 thi- 1 argE3 galK2 ara- 14 xyl-5 mtl tsx33 strA31 supE44 AB2480 as AB1886 but recA 13 AB3072 as AB1886 but recB21 SP263 as AB1886 but tyrA16: :TnlO SP264 asAB1886 but tyrA16: :TnlO recN262 TK907 as AB1886 but recA - 441 sfiA- 11 proA-2 ilv-325 argE + CMll50 as AB1886 but tna- 300::TnlO recF143 CMll37 as AB1886 but proA2 ilv- 325 argE + srl 300: : Tnl O RecA56 All transductions were carried out with P1 cml, clrlO0 (Miller, 1972).
Source or derivation Hill, 1965 P] JC 1 2 3 3 4 x W P 2 uvrA (Southworth and Bridges, 1984) P1 S P 2 6 4 × W P 2 uvrA E.M. Witkin
Howard-Flanders and Theriot, 1966
P1 AB2470 x AB1886 Picksley et al., 1984 Picksley et al., 1984 T. Kato P1 JC12334 x AB1886 P1 JC10240 x TK603
137
Materials and methods
Micro-organisms The strains of Escherichia coli used are listed in Table 1. Media Bacteria were grown with aeration to about 2 x 108/ml in Oxoid nutrient broth No. 2 and plated on the same medium solidified with 1% agar. In some experiments bacteria were grown in the minimal medium of Davis and Mingioli (1950) supplemented with (per litre) 50 mg thymine and either 10 mg tryptophan ( B / r strains) or with 100 mg threonine, leucine, proline, arginine and histidine and 0.25 mg thiamine (K12 strains). As stated in the text, in some experiments the 0.4% glucose was replaced with 0.4% or 0.04% aspartic acid, neutralized with NaOH. Where not specifically stated, incubation temperature was 37°C. Irradiation and experimental procedure Bacteria were suspended in phage buffer (Boyle and Symonds, 1969) at room temperature at a density of around 2 x 108/ml. 8-Methoxypsoralen (Sigma) was added to a final concentration of 20 t t g / m l and the suspension allowed to stand for 5 rain in the dark. Samples were irradiated with UVA 10.5 cm from a Philips 20 W "Blacklight" lamp at a fluence of 2.3 X 10 -4 w / c m 2. After initial exposure a 10 000-fold dilution was made in phage buffer and a further 40-rain exposure to UVA was given. All experiments were carried out at least 3 times with consistent results. Although there appeared to be a considerable day-to-day variation, the ratio in sensitivity between relevant strains remained constant in different experiments.
counted in a liquid scintillation counter. The bacteria were sonicated for 2 × 15 sec and spun at 1 2 0 0 0 X g for 15 rain. The supernatant was counted again in the scintillation counter to determine the recovery of the DNA (usually between 40% and 70%). The DNA content in the samples was determined with the Hoechst Dye procedure (Labarca and Paigen, 1980). DNA content per cell was calculated by dividing the DNA content over the number of initial cells adjusting for the loss made during sedimentation of cell debris after the sonication. Results
Repair in recombination-deficient strains We have tested the effect on crosslink repair of a number of alleles that reduce the ability of the bacteria to perform recombination. When introduced into strain AB1886 the alleles recA56, recB21, recF143 and recN262 all resulted in reduced survival after the induction of crosslinks. The results in strain WP2 uvrA are similar except for the fact that the recB21 allele was not tested in
05
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Estimation of DNA content per cell Bacteria were resuspended in T r i s - E D T A buffer (50 mM Tris, 2 mM EDTA, pH 7.6) and from each culture samples were counted in a bacterial counting chamber. Density was usually around 2 X 108/ml. All further manipulations were done at 0°C. An equal volume of the same medium containing 800 # g / m l freshly dissolved egg-white lysozyme (Sigma) was added and incubated for 10 min to form spheroplasts. Ceils labelled with [3H]thymidine were added and a sample was
Fig. 1. Survival of recombination-deficient derivatives of
Escherichia coli K12 (A) or B / r (B) after the induction of 8-MOP crosslinks (representative experiments). (A) I,, AB1886; O, CM1137 (recA56); D, AB3072 (recB21l); I, CMl150 (reeF143); A, SP264 (recN262). (B) e, WP2 uvrA; O, WP100 (fecAl); I , CMl131 (reeF143); A, CMl145 (recN262). The abscissa represents seconds of exposure to UVA at a fluence of 2.3 x 10 -4 W / m 2 in the presence of 20 /~g/ml 8-MOP. After exposure to UVA 8-MOP was diluted out at least 10 -4 and the samples were exposed to UVA for a further 40 rain to convert as many psoralen adducts as possible to crosslinks. It is assumed that the yield of crosslinks is proportional to the initial UV exposure.
138
this background (Fig. 1). Crosslink repair may be an inducible function and is probably a part of the more general SOS response. It was reported that the recF mutation decreases induction of SOS by ultraviolet light and possibly crosslinks, but not with nalidixic acid (McPartland et al., 1980). Therefore, we have tried to assess whether the sensitivity to crosslinks of CMl131, a derivative of WP2 containing uvrA6 recF143, was the result of a poor induction of the SOS response by pretreating the cells with 50 ~ g / m l nalidixic acid for 60 min at 37°C prior to the induction of crosslinks. It is shown in Fig. 2 that this treatment partly restored the capacity of recF-deficient cells to repair crosslinks, although repair in WP2 uvrA recF + also was stimulated. This suggests that the low number of crosslinks induced in these experiments is in itself not a strong inducer of the SOS response even in rec + cells.
z o kCb OE LL ~D Z
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2
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6
INITIAL UVA DOSE
Fig. 2.Survival of the E. coli B / r strain WP2 uvrA (circles) and reeF143 derivative CMl131 (squares) after induction of crosslinks. The bacteria were either grown in nutrient broth (solid symbols) or treated with 5 0 / ~ g / m l nalidixic acid for 1 h at 37°C (open symbols) before crosslinks were induced as described in the legend to Fig. 1. a
Possible requirement for multiple genomes The requirement for several recombination genes suggested the possible involvement of a recombination event, as is believed to occur during crosslink repair in excision-proficient bacteria. We therefore sought to investigate whether there is a requirement for a second, homologous genome. In the first set of experiments, bacteria were grown for several generations in medium where the main carbon source was aspartic acid neutralized with N a O H , instead of glucose. It has been reported that 0.4% aspartic acid resulted in the presence of only one or slightly more than one genome per cell, conditions in which recombination with another homologous D N A molecule cannot occur (Krasin and Hutchinson, 1977). We could not reproduce this exactly, but reduction of the aspartate concentration in the medium from 0.4% to 0.04% resulted in an increase of the generation time of the bacteria from 1 to 4 h. The average D N A content was reduced from 43.0 _+ 9.5 femto (10 15) g r a m / b a c t e r i u m to 5.6 ± 3.2 fg/bacterium in E. coli WP2 uurA and from 10.4 _+ 2.0 f g / b a c t e r i u m to 5.0 ± 0.9 fg/bacterium in E. coli AB1886. Assuming that the E. coli genome equals 4.6 fg of D N A (Bridges, 1971) this corresponds with a reduction from 9.3 _+ 2.1 to 1.2 ± 0.7 geh o m e s / b a c t e r i u m in WP2 uorA and from 2.3 _+ 0.4 to 1.1 +_ 0.2 genomes/bacterium in AB1886. When the D N A in cells grown on 0.04% aspartic acid was crosslinked with 8-MOP and UVA a higher mortality was observed than in cells grown on glucose or on 0.4% aspartic acid (Fig. 3). This is consistent with a requirement for a second genome, although other trivial explanations cannot be excluded. It may be noted that the hypersensitivity of cells grown on aspartate is in contrast to the general trend that (multigenomic) cells are more resistant to crosslinks in minimal growth medium (Bridges, 1984). The resistant tail in the line representing AB1886 cells grown on 0.04% aspartate medium may reflect a fraction of the cells containing more than one genome. Another approach was to study the survival of crosslinked phage ~ after infection of a uorA-deficient host. We did not find any evidence for crosslink repair when the survival curves were made with a multiplicity of infection (m.o.i.) lower than 1, confirming the results of Bridges and Von
139 ,
,
,
,
,
,
,
,
,
i
r
O5
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001 20
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2
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Fig. 3. Survival of E. coli AB1886 (A) and WP2 uorA (B), after induction of 8-MOP crosslinks (representative experiment), e, bacteria grown in minimal medium with 0.4% glucose; t2, bacteria grown in minimal medium with 0.4% aspartic acid; ©, bacteria grown in minimal medium with 0.04% aspartic acid (resulting in around 1 genome/bacterium). Irradiation procedures and UVA doses are the same as in Fig. 1.
05
Wright (1981). When experiments were done with a m.o.i, of 2 - 4 there was a small but significant increase in survival of incoming ~ phage if the cells contained the recA441 mutation and were grown at 42°C (Fig. 4). When the recA441 host strain was growth at 30°C, the survival of crosslinked ~, phage was identical to the survival in the experiments performed at low m.o.i. The experiments were performed in a strain with the recA441 (formerly tif-1) mutation, as it was reasoned that crosslink repair might be a response induced by the presence of damaged D N A in the bacteria. This could lead to more efficient repair in the bacteria that received a stronger inducing signal, i.e the cells infected with high m.o.i., but in a recA441 mutant the SOS response is induced by a temperature shift from 30°C to 42°C. Moreover, one can assume that the m.o.i, is the same at both temperatures and no corrections have to be made to compensate for the increased possibility of the bacteria to be infected by undamaged particles at high m.o.i. (Hall, 1982). These experiments are consistent with the hypothesis that the presence of at least two genomes in uvrA-deficient bacteria allows more repair of D N A crosslinks.
Discussion Z O V-t.D < r~ LL tD
zo-1 >
0.OS
0.01t
J
150
I
t
300 1,50 INITIAL UVA DOSE
I
600
Fig. 4. Survival of phage 2~ after induction of crosslinks. Phage particles at a density of 109/ml were exposed to UVA in the presence of 20 /~g/ml 8-MOP. After dilution of the 8-MOP and conversion of monoadducts to crosslinks as mentioned in the legend to Fig. 1, the phage were absorbed to E. coli TKg07 (recA441, uorA6) and then plated on a lawn of E. coli AB2480 (recA13, uorA6) to prevent plaque formation by unabsorbed phage. Incubation temperatures were either 30°C (©) or 42°C
(o).,
Cole et al. (1978) have shown that a recA-deficient but excision-proficient strain survives only 0.5-1.6 crosslinks/genome, whereas recB or recF mutants survive between 5.5 and 13 crosslinks/genome. We have found in an exision-deficient background, that recA, recB and recF mutants do not differ significantly from each other in sensitivity to crosslinks. All are more or less equally hypersensitive to crosslinks. It appears that crosslink repair, initiated by the uvrABC endonuclease can be completed via recombination in the absence of either the recB or the recF gene product, whereas in an excision-deficient strain, both these gene products are required for repair. In these strains, the initial step in crosslink repair might be the induction of a single-strand break close to the crosslink by another repair enzyme such as endonuclease III (Radman, 1976; Gates and Linn, 1977) or the recF-dependent protein Z endonuclease (Krivonogov, 1984), followed by a second break in the opposite strand. This may possibly explain the
140
requirement for the recN gene product, which is shown to be associated with repair of DNA double-strand breaks (Lloyd et al., 1983; Picksley et al., 1984). The recB gene product has also been reported to be required for repair of double-strand breaks (Wang and Smith, 1983). It is also possible, notwithstanding, to avoid a double-strand break if single incisions are made in each strand on opposite sides of the crosslink. The free ends thus produced could conceivably initiate crossovers with a homologous duplex on either side of the crosslink. Although there is no evidence on this point, the initial steps of this repair may be the same as those in the repair of psoralen monoadducts, shown to be present in uc'rA-deficient bacteria (Bridges and Stannard, 1982; Bridges, 1983). The role of the recF gene product may be an indirect one, as it is supposed to affect the "specificity of the recA protein binding to different effectors" (Karu and Belk, 1982). A number of observations support this hypothesis. (1) Several mutants that suppress the recF143 phenotype have been mapped in the recA gene (Volkert and Hartke, 1984), (2) the recA441 (formerly tif-1) mutation also suppresses the recF143 defect (Thomas and Lloyd, 1983, Volkert et al., 1984) and concomitantly makes recA protein more efficient in interacting with DNA and nucleoside triphosphates (Phizicky and Roberts, 1981), (3) the induction of the SOS response with nalidixic acid partly restores the capacity of a recF143 uvrA6 strain to repair crosslinks. The fact that recA, recB, recF and recN mutations all block repair of 8-MOP crosslinks in uvrA-deficient bacteria suggests that a recombination step might be involved. Other experiments have produced data consistent with the need for a second, homologous genome in order to carry out such repair. Taken together, these two lines of evidence support a recombinational model, but biochemical evidence will be needed before this can be regarded as established. We are clearly still some way from a functional understanding of crosslink repair in excision-deficient bacteria.
Acknowledgement Marinus Cupido is a recipient of a European Community Fellowship.
References Boyle, J.M., and N. Symonds (1969) Radiation sensitive mutants of T4D. I., T4y: a new radiation sensitive mutant: effect of the mutation on radiation survival, growth and recombination, Mutation Res., 8, 431 439. Bridges, B.A. (1971) RecA+-dependent repair of gamma-ray damage to Escherichia coli does not require recombination between existing homologous chromosomes, J. Bacteriol., 108, 944-945. Bridges, B.A. (1983) Psoralens and serendipity: Aspects of the genetic toxicology of 8-methoxypsoralen, Environ. Mutagen., 5, 329-339. Bridges, B.A. (1984) Further characterization of repair of 8methoxypsoralen crosstinks in UV-excision-defective Escherichia coli, Mutation Res., 132, 153 160. Bridges, B.A., and M. Stannard (1982) A new pathway for repair of cross-linkable 8-methoxypsoralen mono-adducts in uvr strains of Escherichia coli, Mutation Res., 92, 9 14. Bridges, B.A., and A.J. yon Wright (1981) Influence of mutations at the rep gene on survival of Escherichia coli following ultraviolet light irradiation or 8-methoxypsoralen photosensitization, Evidence for a recA + rep+-dependent pathway for repair of DNA crosslinks, Mutation Res., 82, 229-238. Chanet, R., C. Cassier, N. Magaha-Schwenke and E. Moustacchi (1983) Fate of photo-induced 8-methoxypsoralen monoadducts in yeast. Evidence for bypass of these lesions in the absence of excision repair, Mutation Res., 112, 201 214. Cohen, L.F., K.H. Kraemer, H.L. Waters, K.W. Kohn and D.L. Glaubiger (1981) DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation, Mutation Res., 80, 347-356. Cole, R.S. (1970) Light-induced cross-linking of DNA in the presence of a furocoumarin (psoralen); studies with phage lambda, Escherichia coli, and mouse leukemia cells, Biochim. Biophys. Acta, 217, 30-39. Cole, R.S. (1973) Repair of DNA containing interstrand crosslinks in Escherichia coli, Sequential excision and recombination, Proc. Natl. Acad. Sci. (U..S.A.), 70, 1064-1068. Cole, R.S., and D. Zusman (1970) Sedimentation properties of phage DNA molecules containing light-induced psoralen crosslinks, Biochim. Biophys. Acta, 224, 660-662. Cole, R.S., D, Levitan and R.R. Sinden (1976) Removal of psoralen interstrand cross-links from DNA of Escherichia coli, Mechanism and genetic control, J. Mol. Biol., 103, 39-59. Cole, R.S., R.R. Sinden, G.H. Yoakum and S. Broyles (1978) On the mechanism for repair of cross-linked DNA in E. coli treated with psoralen and light, in: P.C. Hanawalt, E.C. Friedberg and C.F. Fox (Eds.), ICN-UCLA Symposia on Molecular and Cellular Biology, DNA Repair Mechanisms, Vol. 9, Academic Press, New York, pp. 287-290. Cupido, M., and B.A. Bridges (1985) Paradoxical behaviour of pKM101; inhibition of uor-independent crosslink repair in Escherichia coli by rnuc gene products, Mutation Res., 145, 49-53.
141 Dall'Acqua, F. (1977) New chemical aspects of the photoreaction between psoralen and DNA, in: A. Castellani (Ed.), Research in Photobiology, Plenum, New York, pp. 245-255. Dall'Acqua, F., S. Marciani and G. Rodrighiero (1970) Interstrand cross-linkages occurring in the photoreaction between psoralens and D N A , FEBS Lett., 9, 121-123. Davis, B.D., and E.S. Mingioli (1950) Mutants of Escherichia coli requiring methionine or vitamin B12, J. Bacteriol., 60, 17-28. Gates, F.T., and S. Linn (1977) Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, nitrous acid, or X-rays, J. Biol. Chem., 252, 2802-2807. Hall,. J.D. (1982) Repair of psoralen-induced crosslinks in cells multiply infected with SV40, Mol. Gen. Genet., 188, 135-138. Hill, R.F. (1965) Ultraviolet-induced lethality and reversion to prototrophy in Escherichia coli strains with normal and reduced dark repair ability, Photochem. Photobiol., 4, 563-568. Howard-Flanders, P., and L. Theriot (1966) Mutants of Escherichia coli K12 defective in DNA repair and in genetic recombination, Genetics, 53, 1137-1150. Karu, A.E., and E.D. Belk (1982) Induction of E. coli recA protein via recBC and alternate pathways: Quantitation by enzyme-linked immunoabsorbent assay (ELISA), Mol. Gen. Genet., 185, 275-282. Krasin, F., and F. Hutchinson (1977) Repair of DNA doublestrand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome, J. Mol. Biol., 116, 81-98. Krivonogov, S.V. (1984) The recF-dependent endonuclease from Escherichia coli K12, Formation and resolution of pBR322 DNA multimers, Mol. Gen. Genet., 196, 105-109. Kuhn, B., M~ Abdel-Monem and H. Hoffmann-Berling (1979) DNA helicases, Cold Spring Harbor Symp. Quant. Biol., 43, 63-67. Kumura, K., K. Oeda, M. Akiyama, T. Horiuchi and M. Sekiguchi (1983) The uvrD gene of E. coli: Molecular cloning and expression, in: E.C. Friedberg and B.A. Bridges (Eds.), UCLA Symposia on Molecular and Cellular Biology, Cellular Responses to DNA Damage, Vol. 11, Liss, New York, pp. 51-62. Labarca, C., and K. Paigen (1980) A simple, rapid and sensitive DNA assay procedure, Anal. Biochem., 102, 344-352. Little, J.W., and D. Mount (1982) The SOS regulatory system of Escherichia coli, Cell, 29, 11-22. Lloyd, R.G., S.M. Picksley and C. Prescott (1983) Inducible expression of a gene specific for the recF pathway for recombination in Escherichia coli K12, Mol. Gen. Genet., 190, 162-167. McPartland, A., L. Green and H. Echols (1980) Control of recA gene RNA in E. coli: Regulatory and signal genes, Cell, 20, 731-737.
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, p. 203. Phizicky, E.M., and J.W. Roberts (1981) Induction of SOS functions: regulation of proteolytic activity of E. coli Rec protein by interaction with D N A and nucleoside triphosphate, Cell, 25, 259-267. Picksley, S.M., P.V. Attfield and R.G. Lloyd (1984) Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product, Mol. Gen. Genet., 195, 267-274. Piette, J.G., and J.E. Hearst (1983) Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5540-5544. Radman, M. (1975) SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, in: P. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, part A, Plenum, New York, pp. 355-367. Radman, M. (1976) An endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA, J. Biol. Chem., 251, 1438-1445. Roberts, J., and R. Devoret (1983) Lysogenic induction, in: Lambda 2, Cold Spring Harbor Laboratory, New York, pp. 123-144. Sancar, A., and W.D. Rupp (1983) A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33, 249-260. Sinden, R.R., and R.S. Cole (1978) Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: Effects of mutations influencing genetic recombination and DNA metabolism, J. Bacteriol., 136, 538-547. Southworth, M.W., and B.A. Bridges (1984) Influence of reeF on spontaneous mutation in Escherichia coli, Mutation Res., 140, 67-69. Thomas, A., and R.G. Lloyd (1983) Control of recA dependent activities in Escherichia coli: a possible role for the recF product, J. Gen. Microbiol., 129, 681-686. Volkert, M.R., and M.A. Hartke (1984) Suppression of Escherichia coli recF mutations by recA-linked srfA mutations, J. Bacteriol., 156, 1093-1098. Volkert, M.R., L.J. Margossian anbd A.J. Clark (1984) Two component suppression of recF143 by recA441 in Escherichia coli K12, J. Bacteriol., 160, 702-705. Wang, T.V., and K.C. Smith (1983) Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB, J. Bacteriol., 156, 1093-1098. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.