J. Photochem.
Photobiol.
B: Biol.,
IO (1991)
329-337
329
uvr&dependent, recF-independent post-replication (or replication) repair in Eschwichia coli V. Slezkikovh
and M. SedIiakovA
Cancer
Research Institute, Slovak Academy Genetics, Cs. armady 21, 812 32 Bratislava
of Sciences, Department (Czechoslovakia)
of Molecular
(Received November 14, 1990; accepted February 25, 1991)
Keywords.
Post-replication
repair, recF cells, uvrB
cells, dimer excision.
Abstract In UV-damaged cells, a large fraction of pyrimidine dimers may remain unexcised and may be tolerated by a uvrB recA Leti-dependent non-excisional mode of repair (M. SedIiakova, J. Brozmanova, F. M&ek and K. Kleibl, Btiphys. J., 36 (1981) 429-441). We show here that a similar repair pathway operates in the Escherichia coli recF 143 single mutant but not in the recF uvrB double mutant. This indicates that the putative repair pathway is recF independent.
1. Introduction In wild-type Escherichia co& the bulk of W-induced pyrimidine dimers are removed by the uvrABC excision nuclease [l] which distinguishes conformational changes of DNA molecules rather than dimers themselves [ 2-41. The regulation of the genes which govern the synthesis of this enzyme is surprisingly complex [ 5-81, especially with respect to its uvrB component [ 7, 91. The involvement of the uvrB gene in the regulation of DNA replication has been suggested [lo]. This, as well as earlier data indicating that the uvrB [ 11, 121 and uvrD [ 13, 141 genes can, in some respect, complement the polA deficiency, has led to the prediction of a multifunctional role of the uvr genes in DNA repair and replication [15, 161. In accord with the above-mentioned findings, in vivo experiments revealed that the uvrB gene is involved in a mode of post-replication repair [ 17, 131. It has also been shown that, in cells irradiated with two separate W fluences, and/or predamaged by thymine starvation, a large fraction of the W-induced dimers may remain unexcised and may be tolerated by a uvrB recA Lzz4-dependent non-excisional mode of repair [ 1g-221. The molecular mechanism of the putative uvr-dependent, non-excisional repair pathway, which is apparently damage inducible, is obscure. In this paper, we have tried to determine whether it requires a functional recF gene,
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Elsevier Sequoia, Lausanne
330
known to be involved in an inducible pathway of DNA redombination [23, 241 as well as in SOS regulation [25, 261. Our data indicate that the putative repair pathway is recF independent.
2. Materials
and methods
2.1. Bacterial strains, cultivation and irradiation The bacterial strains used were derivatives of E. coli K-l 2. The strain JC 9239 was recF 143 his pro arg thr leu thi, kindly provided by S. M. Picksley (Genetic Department, University of Nottingham, Nottingham N67 2RD, U.K.). Its thymine auxotroph was selected by the trimethoprim method (271. The strain JC 8911 recF 143 uvrB, thy A18 thr leu arg pro his thi was a gift of A. J. Clark (Department of Molecular Biology, University of California, Berkeley, CA 94720, U.S.A.). Cells were cultured in glucose-salt medium with 2 pg ml-’ of thiamine, supplemented with 0.3% casamino acids and, when required, with 2 pg ml-’ of thymine [28]. The medium not supplemented with casamino acids and thymine is designated as ‘minimal’; the supplemented medium is called ‘complete’. In all experiments cells were cultivated in complete medium up to a density of approximately 1 X 10’ cells ml-‘, harvested by filtration and transferred into minimal medium. 2.2. Inhibition of dimer excision by pretreatment with cytidine The changes produced by thymine starvation in DNA molecules lead to cell death [29] which may be prevented by a simultaneous starvation of essential amino acids [30]. The influence of thymine-less, ammo acid-less pretreatment on cell viability, UV resistance and dimer excision has been studied in various strains of E. coli [ 3 11. It has been shown that the effect of this pretreatment depends on the intracellular pool of thymine. In the strain E. coli 15 with a small pool of thymine [ 321, this pretreatment efficiently inhibits dimer excision but does not completely prevent thymine-less death [ 3 11. In E. coli K-l 2 cells, with a large pool of thymine [ 321, this pretreatment completely eliminates thymine-less death but does not inhibit dimer excision [ 311. However, inhibition of dimer excision (and/or elimination of thymineless death) can be achieved by the adjustment of the ratio of the thymine to amino acid pool [31]_ The depletion of thymine during preirradiation starvation for thymine and ammo acids may be accelerated by the addition of cytidine, which is known to prevent conversion of thymine into thymidine [33] by a mechanism described earlier [ 341. It has been shown that UV pretreatment with cytidine inhibits dimer excision in E. co,% B/r thy trp. When thymine-less death is prevented by concomitant inhibition of protein synthesis (i.e. when cells are preincubated in thy- trp- medium containing cytidine), cell survival after UV irradiation is slightly increased in spite of the inhibition of dimer excision
1201.
331
In these experiments, cells were cultivated in minimal medium (without thymine and casamino acids) with 50 pg ml-’ of cytidine for 90 min prior to UV irradiation. 2.3. W irradiation Cells were irradiated in petri dishes in a 1 mm deep, manually stirred suspension. A germicidal Philips TUV 15 W lamp was used as the UV light source. The intensity of radiation, measured by an IL 254 germicidal photometer (International Light, Newbury-Port, MA, U.S.A.) was 0.633 J rnw2 s-l. 2.4. Thymine dimer estimation The thymine dimer content was determined by two-dimensional radiochromatography as described by Carrier and Setlow [35].
paper
2.5. Molecular weight determination Sedimentation patterns of DNA were determined in alkaline sucrose gradients by the method of McGrath and Williams [ 361. At the indicated time after irradiation (see Pig. 3, later), cells were pulse labelled with 3Hthymidine (3.7 MBq ml-‘; specific activity, 960 GBq mmol-‘) for 10 mm. The lysis procedure and DNA sedimentation were performed as described in ref. 21. 2.4. Determination of endmuclease-sensitive sites An in vitro system described by Wilkins [ 371 was used. UV endonuclease, isolated from cells of Micrococcus lziteus according to the method of Carrier and Setlow [38], was kindly provided by Dr. Kleibl from our institute.
3. Results 3.1. Dimm excision As shown in Pig. 1, the E. coli recF 143 cells efficiently excised dimers when irradiated in the exponential phase of growth. However, if they were incubated with cytidine prior to UV irradiation, about one-half of the original amount of dimers remained unexcised. This indicates that the recF 143 mutant behaves like the wild-type E. coli previously described [20, 211. 3.2. Cell survival As shown in Fig. 2, the cell survival was similar in recF 143 cells in which efficient dimer excision occurred (exponential growth) and in which one-half of the dimers were left unexcised (preincubation in thymine and casamino acids lacking medium containing cytidine). However, both UWB single mutant and uvrB recF double mutant cells were extremely UV sensitive. 3.3. Sedimentation properties of daughter DNA In further experiments, we compared the sedimentation properties of DNA synthesized in both recF 143 and uvrB recF 143 cells on templates
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130
MIN AFTER UV Fig. 1. Depression of pyrimidine diier excision in UV-irradiated recF 143 cells pretreated with @dine. Cells were cultivated in complete medium supplemented with 18.5 kBq ml-’ thymine-2-r% (spechic activity, 1850 MBq mM_r) up to a density of about 1 X 10’ cells ml-‘. Subsequently, they were filtered in minimal medium and divided into two aliquots. One was irradiated (50 J m-‘) and supplemented with casamino acids and thymine (0); the other was cultivated for 90 min with &dine (50 pg ml-‘) and then irradiated and supplemented as above (e). At the indicated time intervals, samples were taken for dimer estimation.
containing similar amounts of unexcised dimers. To achieve this, the reck 143 cells were pretreated with cytidine, which caused about one-half of the dimers to remain unexcised. They were then exposed to 20 or 50 J m-‘, whereas the reck uvrB cells were irradiated with 10 J mV2. As shown in Figs. 3(A) and 3(C), DNA synthesized during the 10 mm pulse labelling immediately after irradiation in the reck 143 single mutant sedimented in two regions of the gradient. The high-molecule-weight DNA corresponds to repair synthesis, whereas the DNA sedimenting in the low-molecular-weight peak should represent the short fragments of daughter DNA. As shown in Fig. 3(D), the DNA synthesized 120 min after irradiation with 20 J mP2 in recF cells was of normal size; after the dose of 50 J mV2 some shift to
333
10
20
20 13
I 10
50
Fig. 2. Survival as a function of UV dose. Untreated cells were grown in complete medium up to a density of 1 X lo8 ml-‘, filtered, washed, resuspended in minimal medium and irradiated. Pretreated cells at the same density were transferred into minimal medium with cytidine (50 pg ml-‘) and incubated for 90 min. They were then filtered, washed, resuspended in minimal medium and irradiated. 0, AB 1157 untreated: 0, recF 143 untreated; 0, recF 143 treated; V uvrB, untreated; A, recF 143 uvrB, untreated.
higher molecular weight was registered (Fig. 3(F)). In reck uvrB cells only short DNA fragments were synthesized at that time (Fig. 3(E)). Finally, DNA synthesized in cytidine-pretreated reck cells, as well as in exponentially growing reck uvrB cells, was exposed to endonuclease from Micrococcus 1uteu.s to determine whether it contained endonuclease-sensitive sites. Sedimentation analysis showed that both were free of such sites (Figs. 4(A) and 4(B)).
4. Discussion It has previously been shown that, in UV-irradiated E. coli prestarved for thymine, a large fraction of the dimers remain unexcised and are tolerated on replication since they can be detected in replicated DNA [ 2 11. A similar phenomenon is observed in cells predamaged with a low UV dose [ 191. uvrB, cells, unlike wild-type cells, cannot survive and restore DNA synthesis with as many dimers in their template. It is therefore concluded that the
334
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profiles of the newly synthesized, pulse-labelled DNA after cytidiie pretreatment in recF 143 and untreated uvr& recF 143 cells. Cytidine-treated recF 143 cells (as described in Fig. 1) were irradiated with a dose of 20 (A, D) or 50 J mm2 (C, F). Untreated ~~23~ recF 143 cells were irradiated with a dose of 10 J m-’ (B, E). At the indicated times after irradiation, cells were pulse labelled for 10 mm with 40 MBq ml-’ 6-3H-thymidine (spccitic activity, 960 GBq mmol-I). After cooling they were lysed, DNA denaturated and analysed in alkaline sucrose gradients. Arrows indicate the sedimentation of unirradiated, pulse-labelled DNA. Direction of sedimentation is from right to left.
Fig. 3.
Sedimentation
uvrB gene fulfils a further role in DNA repair and is involved not only in excision but also in toleration of unexcised dimers [ 191. The data presented in this paper indicate that prestarvation for thymine mediated by incubation with cytidine (see Section 2) causes an inhibition of dimer excision in E. coli recF 143 cells (Fig. 1) similar to that observed previously in wild-type cells [ 20, 211. Similarly, no decrease in survival of recF 143 cells is observed when they contain unexcised dimers, whereas the reck uvrB double mutant is extremely UV sensitive (Fig. 2). Dimers remaining unexcised are not an obstacle for the synthesis of normal size DNA in the recF 143 single mutant, but they are in the recF uvrB double mutant (Fig. 3). We did not measure the effect of cytidine pretreatment on the removal of the [6-41 photoproduct, which is the second major photoproduct in the DNA of UV-irradiated cells [39-411. From the observation that the purified uvrABC enzymes cleave both the [6-41 photoproduct and the cyclobutane dimer in the same fashion [ 11, we can assume that the removal of the two photoproducts is affected to the same degree. However, since the amount of [6-41 photoproduct is only about 7%-15% of the number of cyclobutane dimers [40, 411 and its lethal effect is no greater than that of the latter [42], we believe that the presence (or absence) of this photoproduct in the
335
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profiles of pulse-labelled DNA from UV-irradiated, cytidine-pretreated Fig. 4. Sedimentation recF (A) and UV-irradiated, untreated recF uvrB (B) cells with (-) or without (. . . .) endonuclease treatment. Cells were cultured in complete medium up to a density of about 1 X IO* ml-‘, filtered, washed and resuspended in minimal medium. recF cells were subsequently cultured in the presence of 50 pg ml-’ of @dine for 90 min, filtered, washed and, after resuspending in minimal medium, irradiated with a dose of 20 J m-‘. After 110 mm cultivation in complete medium they were pulse labelled with 3H-thymidine for 10 mm. Untreated recF uvrB cells were irradiated with a dose of 10 J mm2 and, after 110 mm incubation, were pulse labellcd as above. After turning into spheroplasts, samples of each culture were divided into two aliquots, one of which was treated with endonuclease, and both were analysed in alkaline sucrose gradients. Sedimentation is from right to left.
DNA of cytidine-treated cells will not substantially influence our results. Our data suggest that the non-excisional uvr-dependent repair pathway described for wild-type cells operates in E. coli recF 143 cells, which means that the putative pathway is reck independent. The molecular mechanism of this recA &zA uvrB-dependent pathway is unknown. It has been speculated that it may be mediated by a ‘copy choice bypass’ of a lesion, where information is taken from a daughter strand of a sister duplex instead of the parental strand containing the lesion [ 191. Such an error-free bypass, mediated by the formation of a recA protein three-stranded structure and involving a breaking and rejoining step, has also been considered by other workers [43, 441.
References 1 A. Sancar and W. D. Rupp, A novel DNA repair enzyme: uvrABC excision nuclcase of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33 (1983) 249-260. 2 A. T. Yeung, B. K. Jones, M. Capano and T. Chu, The repair of psoralen monoadducts by the uvrABC endonuclease, Nucleic Acids Res., 15 (1987) 4857-4971.
336 3 A. Sancar, K. A. Franklin, G. B. Sancar and M. S. Tang, Repair of psoralen and acetylamlnoiluorene DNA adducts by ABC excinuclease, J. Mol. Bid., 184 (1985) 725-734. 4 E. Seeberg, A. L. Steinum, M. Nordenskiold, S. Soderthall and B. Jcnstrom, Strand-break formation in DNA modified by benzo(a)pyrene diolepoxide. Quantitative cleavage by Escherichia coli uvrABC endonuclcase, Mutat. Res., 112 (1983) 139-145. 5 M. Fogliano and P. F. Shendel, Evidence for the inducibility of the uvrB operon, Nature, 289 (1981) 196-198. 6 C. J. Kenyon and G. C. Walker, Expression of the Escherichia coli uvrA gene is inducible, Nature, 289 (1981) 808-810. 7 G. B. Sancar, A. Sancar, J. W. Little and W. D. Rupp, The uvrB gene of Escherichia coli has both led-repressed and led-independent promotors, Cell, 28 (1982) 523-530. 8 A. Sancar, G. B. Sancar, W. D. Rupp, J. W. Little and D. W. Mount, LexA protein inhibits transcription of the Escherichia coli uvrA gene in vitro, Nature, 298 (1983) 96-98. 9 E. A. van den Berg, R. H. Geerse, J. Memelink, R. A. L. Bovenberg, F. A. Magnet and P. van de Putte, Analysis of regulatory sequences upstream of the Escherichia coli uvrB gene; involvement of the Dna.A protein, Nucleic Acids Res., 13 (1985) 1829-1840. 10 R. S. Fuller, B. E. Funnel and A. Komberg, The DnuA protein complex with the Eschenkhia coli chromosomal replication origin (orlC) and other DNA sites, Cell, 38 (1984) 889-900. 11 H. Shlzuya and D. Dykhuizen, Conditional lethality of deletions which include uvrB in stralns of Escherichia coli lacking deoxyribonucleic acid polymerasc I, J. Bacteriol., 112
(1972) 676-681. 12 M. Morimyo and Y. Shimazu, Evidence that the gene uvrB is indispensable for polymerase I deficient strain of Escherichia coli K-12, Mol. Gen. Genet., 147 (1976) 243-250. 13 T. Horiuchi and T. Nagata, Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymcrase I deficiency, Mol. Gen. Genet., I23 (1973) 89-110. 14 G. B. Smirnov, E. V. Fllkova, A. G. Skavronskaya, A. S. Saenko and B. I. Slnzlnis, Loss and restoration of viability of Escherichia coli due to combination of mutations affecting DNA polymerase I and repair activities, Mol. Gen. Genet., 121 (1973) 139-150. 15 E. C. Frledbcrg, DNA Repair, Freeman, New York, 1985, p. 234. 16 E. C. Friedbcrg, The molecular biology of nucleotide excision repair of DNA: recent progress, in A. Collins, R. T. Johnson and J. M. Boyle (cds.), Molecular biology and DNA repair, J. Cell Sci., SuppL. 6 (1987) l-23. 17 A. K. Ganesan and P. C. Seawell, The effect of ZexA and recF mutations on postreplication repair and DNA synthesis ln Escherichia coli K-12, Mol. Gen. Genet., 141 (1975) 189-206. 18 R. H. Rothman and A. J. Clark, The dependence of postrcplication repair on uvrB in a recF mutant of Escherichia coli K-12, Mol. Gen. Genet., 155 (1977) 279-286. 19 M. Sedliakova, J. Brozmanova, F. Mdek and K. Kleibl, Evidence that dlmers remaining in preinduced Escherkhiu coli B/r Her+ become insensitive after DNA replication to the extract from Micrococcus Luteus, Biophys. J., 36 (1981) 429-441. 20 J. Brozmanova, L. M&kova and M. Sedliakova, Inhibition of thymlne dimer excision after preirradiation inhibition of DNA synthesis by cytidlnc, Stud. Biophys., 36/37 (1973) 245-252. 21 M. Sedliakova, J. Brozmanova, F. M&ek and V. Slezarikova, Interaction of restoration processes lnUVirradiatedEscherichiacolicells,Photochem. Photobiol., 25 (1977) 259-264. 22 M. Sedliakova, V. Slezarikova, F. M&ek and J. Brozmanova, W-inducible repair: influence on survival, dlmer excision, DNA replication and breakdown in Escherichia coli B/r Her’ cells, Mol. Gen. Genet., 160 (1978) 81-87. 23 Z. I. Horll and A. J. Clark, Genetic analysis of the recF pathway of genetic recombination ln Escherichia coli K-12: isolation and characterization of mutants, J. Mol. BioL., 80 (1973) 327-344. 24 S. M. Picksley, R. G. Lloyd and C. Buckman, Genetic analysis and regulation of inducible recombination in Escherichia coli K-12, Cold Spring Harbor Symp. Quant. Biol., 49
(1984) 469-474. 25 B. Thorns and W. Wackemagel, Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant, J. Bacterial., 169 (1987) 1731-1736.
337 26 A. McPartland,
L. Green and H. Echols, Control of recA regulatory and signal genes, Cell, 20 (1980) 731-737. 27 J. H. Miller, Selection of thystrains with trimethoprim, 28 29 30 31
32 33
gene
RNA ln Escherichia
in Experiments
coli:
in Mobcular
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, pp. 218-220. V. Slezarikova, F. Ma&k and M. Sedliakova, Depression of dimer excision in ret _ UUT + mutant of Escherichia coli K-12, Stud. Biophys., 44 (1974) 1-6. S. S. Cohen and H. D. Bamer, Studies of unbalanced growth in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A., 40 (1954) 885-893. 0. Maaloe and P. C. Hanawalt, Thymine deficiency and the normal DNA replication cycle. I, J. Mol. Biol., 3 (1961) 144-155. M. Sedliakova, F. MaSek, J. Brozmanova, L. Mdkova and V. Slezkikova, Depression of thymine dimer excision in various excision-proficient strains of Escherichia coli, Biochim. Biophys. Acta, 349 (1974) 442-446. L. G. Care and C. M. Berg, Chromosome replication in some strains of Escherichia coli K-12, Cold Spring Harbor Symp. Quant. Biol., 33 (1968) 559-573. D. Frelfelder, Technique for starvation of Escherichia coli of thymine, J. Bacterial., 90
(1965) 1153-l 154. 34 R. Mantsavinos and S. Zamenhof, Pathway for the biosynthesis of thymidilic acid in bacterial mutants, J. Biol. Chem, 236 (1961) 876-882. 35 W. L. Carrier and R. B. Setlow, The excision of pyrlmidine dimers, in L. Grosman and K. Moldave (eds.), Methods in Enzymology, Vol. XXI, Academic Press, New York, 1971, pp. 230-237. 36 R. McGrath and R. W. Wiiiams, Reconstruction in viva of irradiated Eschenfchia coli deoxyribonucleic acid: the rejoining of broken pieces, Nature, 212 (1966) 534-535. 37 R. J. Wilkins, Endonuclease-sensitive sites in the DNA of irradiated bacteria: a rapid and sensitive assay, Biochim. Biophys. Acta, 312 (1973) 33-37. 38 W. L. Carrier and R. B. Sctlow, Endonuclease from Micrococcus luteus which has activity toward ultraviolet-irradiated deoxyribonucleic acid. Purification and properties, J. Bacterial.,
IO2 (1970) 178-186. 39 M. H. Patrick, Near-UV photolysis of pyrimidine hetero adducts in Escherichia coli DNA, Photo&em. Photobiol., 11 (1970) 477-485. 40 M. H. Patrick, Studies on thymine-derived photoproducts in DNA. I. Formation and biological role of pyrimidlne adducts in DNA, Photo&em. Photobiol., 25 (1977) 357-372. 41 W. A. Franklin and W. A. Haseltine, Removal of UV light-induced pyrimidine-pyrlmidone [6-4] products from Escherichia coli DNA requires the uvrA, uvrB and uvrC gene products, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3821-3824. 42 M.-S. Thang, J. Hmclr, D. Mitchell, J. Ross and J. Clarkson, The relative cytotoxicity and mutagenicity of cyclobutanc pyrlmidlne dimers and 16-41 photoproducts in Escherichia coli cells, Mutat. Res., 161 (1986) 9-17. 43 H. Eschols, Mutation rate: some biological and biochemical considerations, Biochimie, 64 (1982) 571-575. 44 R. Woodgate, M. Rsjagopalan, Ch. Lu and H. Echols, Escherichia coli: puri6cation and interaction with Urn@ Sci. U.S.A., 86 (1989) 7301-7305.
UmuC mutagenesis protein of and UmuD’, Proc. Natl. Acad.
Photobiol.
B: Biol.,
IO (1991)
329-337
329
uvr&dependent, recF-independent post-replication (or replication) repair in Eschwichia coli V. Slezkikovh
and M. SedIiakovA
Cancer
Research Institute, Slovak Academy Genetics, Cs. armady 21, 812 32 Bratislava
of Sciences, Department (Czechoslovakia)
of Molecular
(Received November 14, 1990; accepted February 25, 1991)
Keywords.
Post-replication
repair, recF cells, uvrB
cells, dimer excision.
Abstract In UV-damaged cells, a large fraction of pyrimidine dimers may remain unexcised and may be tolerated by a uvrB recA Leti-dependent non-excisional mode of repair (M. SedIiakova, J. Brozmanova, F. M&ek and K. Kleibl, Btiphys. J., 36 (1981) 429-441). We show here that a similar repair pathway operates in the Escherichia coli recF 143 single mutant but not in the recF uvrB double mutant. This indicates that the putative repair pathway is recF independent.
1. Introduction In wild-type Escherichia co& the bulk of W-induced pyrimidine dimers are removed by the uvrABC excision nuclease [l] which distinguishes conformational changes of DNA molecules rather than dimers themselves [ 2-41. The regulation of the genes which govern the synthesis of this enzyme is surprisingly complex [ 5-81, especially with respect to its uvrB component [ 7, 91. The involvement of the uvrB gene in the regulation of DNA replication has been suggested [lo]. This, as well as earlier data indicating that the uvrB [ 11, 121 and uvrD [ 13, 141 genes can, in some respect, complement the polA deficiency, has led to the prediction of a multifunctional role of the uvr genes in DNA repair and replication [15, 161. In accord with the above-mentioned findings, in vivo experiments revealed that the uvrB gene is involved in a mode of post-replication repair [ 17, 131. It has also been shown that, in cells irradiated with two separate W fluences, and/or predamaged by thymine starvation, a large fraction of the W-induced dimers may remain unexcised and may be tolerated by a uvrB recA Lzz4-dependent non-excisional mode of repair [ 1g-221. The molecular mechanism of the putative uvr-dependent, non-excisional repair pathway, which is apparently damage inducible, is obscure. In this paper, we have tried to determine whether it requires a functional recF gene,
loll-1344/91/$3.50
0 1991 -
Elsevier Sequoia, Lausanne
330
known to be involved in an inducible pathway of DNA redombination [23, 241 as well as in SOS regulation [25, 261. Our data indicate that the putative repair pathway is recF independent.
2. Materials
and methods
2.1. Bacterial strains, cultivation and irradiation The bacterial strains used were derivatives of E. coli K-l 2. The strain JC 9239 was recF 143 his pro arg thr leu thi, kindly provided by S. M. Picksley (Genetic Department, University of Nottingham, Nottingham N67 2RD, U.K.). Its thymine auxotroph was selected by the trimethoprim method (271. The strain JC 8911 recF 143 uvrB, thy A18 thr leu arg pro his thi was a gift of A. J. Clark (Department of Molecular Biology, University of California, Berkeley, CA 94720, U.S.A.). Cells were cultured in glucose-salt medium with 2 pg ml-’ of thiamine, supplemented with 0.3% casamino acids and, when required, with 2 pg ml-’ of thymine [28]. The medium not supplemented with casamino acids and thymine is designated as ‘minimal’; the supplemented medium is called ‘complete’. In all experiments cells were cultivated in complete medium up to a density of approximately 1 X 10’ cells ml-‘, harvested by filtration and transferred into minimal medium. 2.2. Inhibition of dimer excision by pretreatment with cytidine The changes produced by thymine starvation in DNA molecules lead to cell death [29] which may be prevented by a simultaneous starvation of essential amino acids [30]. The influence of thymine-less, ammo acid-less pretreatment on cell viability, UV resistance and dimer excision has been studied in various strains of E. coli [ 3 11. It has been shown that the effect of this pretreatment depends on the intracellular pool of thymine. In the strain E. coli 15 with a small pool of thymine [ 321, this pretreatment efficiently inhibits dimer excision but does not completely prevent thymine-less death [ 3 11. In E. coli K-l 2 cells, with a large pool of thymine [ 321, this pretreatment completely eliminates thymine-less death but does not inhibit dimer excision [ 311. However, inhibition of dimer excision (and/or elimination of thymineless death) can be achieved by the adjustment of the ratio of the thymine to amino acid pool [31]_ The depletion of thymine during preirradiation starvation for thymine and ammo acids may be accelerated by the addition of cytidine, which is known to prevent conversion of thymine into thymidine [33] by a mechanism described earlier [ 341. It has been shown that UV pretreatment with cytidine inhibits dimer excision in E. co,% B/r thy trp. When thymine-less death is prevented by concomitant inhibition of protein synthesis (i.e. when cells are preincubated in thy- trp- medium containing cytidine), cell survival after UV irradiation is slightly increased in spite of the inhibition of dimer excision
1201.
331
In these experiments, cells were cultivated in minimal medium (without thymine and casamino acids) with 50 pg ml-’ of cytidine for 90 min prior to UV irradiation. 2.3. W irradiation Cells were irradiated in petri dishes in a 1 mm deep, manually stirred suspension. A germicidal Philips TUV 15 W lamp was used as the UV light source. The intensity of radiation, measured by an IL 254 germicidal photometer (International Light, Newbury-Port, MA, U.S.A.) was 0.633 J rnw2 s-l. 2.4. Thymine dimer estimation The thymine dimer content was determined by two-dimensional radiochromatography as described by Carrier and Setlow [35].
paper
2.5. Molecular weight determination Sedimentation patterns of DNA were determined in alkaline sucrose gradients by the method of McGrath and Williams [ 361. At the indicated time after irradiation (see Pig. 3, later), cells were pulse labelled with 3Hthymidine (3.7 MBq ml-‘; specific activity, 960 GBq mmol-‘) for 10 mm. The lysis procedure and DNA sedimentation were performed as described in ref. 21. 2.4. Determination of endmuclease-sensitive sites An in vitro system described by Wilkins [ 371 was used. UV endonuclease, isolated from cells of Micrococcus lziteus according to the method of Carrier and Setlow [38], was kindly provided by Dr. Kleibl from our institute.
3. Results 3.1. Dimm excision As shown in Pig. 1, the E. coli recF 143 cells efficiently excised dimers when irradiated in the exponential phase of growth. However, if they were incubated with cytidine prior to UV irradiation, about one-half of the original amount of dimers remained unexcised. This indicates that the recF 143 mutant behaves like the wild-type E. coli previously described [20, 211. 3.2. Cell survival As shown in Fig. 2, the cell survival was similar in recF 143 cells in which efficient dimer excision occurred (exponential growth) and in which one-half of the dimers were left unexcised (preincubation in thymine and casamino acids lacking medium containing cytidine). However, both UWB single mutant and uvrB recF double mutant cells were extremely UV sensitive. 3.3. Sedimentation properties of daughter DNA In further experiments, we compared the sedimentation properties of DNA synthesized in both recF 143 and uvrB recF 143 cells on templates
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100
130
MIN AFTER UV Fig. 1. Depression of pyrimidine diier excision in UV-irradiated recF 143 cells pretreated with @dine. Cells were cultivated in complete medium supplemented with 18.5 kBq ml-’ thymine-2-r% (spechic activity, 1850 MBq mM_r) up to a density of about 1 X 10’ cells ml-‘. Subsequently, they were filtered in minimal medium and divided into two aliquots. One was irradiated (50 J m-‘) and supplemented with casamino acids and thymine (0); the other was cultivated for 90 min with &dine (50 pg ml-‘) and then irradiated and supplemented as above (e). At the indicated time intervals, samples were taken for dimer estimation.
containing similar amounts of unexcised dimers. To achieve this, the reck 143 cells were pretreated with cytidine, which caused about one-half of the dimers to remain unexcised. They were then exposed to 20 or 50 J m-‘, whereas the reck uvrB cells were irradiated with 10 J mV2. As shown in Figs. 3(A) and 3(C), DNA synthesized during the 10 mm pulse labelling immediately after irradiation in the reck 143 single mutant sedimented in two regions of the gradient. The high-molecule-weight DNA corresponds to repair synthesis, whereas the DNA sedimenting in the low-molecular-weight peak should represent the short fragments of daughter DNA. As shown in Fig. 3(D), the DNA synthesized 120 min after irradiation with 20 J mP2 in recF cells was of normal size; after the dose of 50 J mV2 some shift to
333
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Fig. 2. Survival as a function of UV dose. Untreated cells were grown in complete medium up to a density of 1 X lo8 ml-‘, filtered, washed, resuspended in minimal medium and irradiated. Pretreated cells at the same density were transferred into minimal medium with cytidine (50 pg ml-‘) and incubated for 90 min. They were then filtered, washed, resuspended in minimal medium and irradiated. 0, AB 1157 untreated: 0, recF 143 untreated; 0, recF 143 treated; V uvrB, untreated; A, recF 143 uvrB, untreated.
higher molecular weight was registered (Fig. 3(F)). In reck uvrB cells only short DNA fragments were synthesized at that time (Fig. 3(E)). Finally, DNA synthesized in cytidine-pretreated reck cells, as well as in exponentially growing reck uvrB cells, was exposed to endonuclease from Micrococcus 1uteu.s to determine whether it contained endonuclease-sensitive sites. Sedimentation analysis showed that both were free of such sites (Figs. 4(A) and 4(B)).
4. Discussion It has previously been shown that, in UV-irradiated E. coli prestarved for thymine, a large fraction of the dimers remain unexcised and are tolerated on replication since they can be detected in replicated DNA [ 2 11. A similar phenomenon is observed in cells predamaged with a low UV dose [ 191. uvrB, cells, unlike wild-type cells, cannot survive and restore DNA synthesis with as many dimers in their template. It is therefore concluded that the
334
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profiles of the newly synthesized, pulse-labelled DNA after cytidiie pretreatment in recF 143 and untreated uvr& recF 143 cells. Cytidine-treated recF 143 cells (as described in Fig. 1) were irradiated with a dose of 20 (A, D) or 50 J mm2 (C, F). Untreated ~~23~ recF 143 cells were irradiated with a dose of 10 J m-’ (B, E). At the indicated times after irradiation, cells were pulse labelled for 10 mm with 40 MBq ml-’ 6-3H-thymidine (spccitic activity, 960 GBq mmol-I). After cooling they were lysed, DNA denaturated and analysed in alkaline sucrose gradients. Arrows indicate the sedimentation of unirradiated, pulse-labelled DNA. Direction of sedimentation is from right to left.
Fig. 3.
Sedimentation
uvrB gene fulfils a further role in DNA repair and is involved not only in excision but also in toleration of unexcised dimers [ 191. The data presented in this paper indicate that prestarvation for thymine mediated by incubation with cytidine (see Section 2) causes an inhibition of dimer excision in E. coli recF 143 cells (Fig. 1) similar to that observed previously in wild-type cells [ 20, 211. Similarly, no decrease in survival of recF 143 cells is observed when they contain unexcised dimers, whereas the reck uvrB double mutant is extremely UV sensitive (Fig. 2). Dimers remaining unexcised are not an obstacle for the synthesis of normal size DNA in the recF 143 single mutant, but they are in the recF uvrB double mutant (Fig. 3). We did not measure the effect of cytidine pretreatment on the removal of the [6-41 photoproduct, which is the second major photoproduct in the DNA of UV-irradiated cells [39-411. From the observation that the purified uvrABC enzymes cleave both the [6-41 photoproduct and the cyclobutane dimer in the same fashion [ 11, we can assume that the removal of the two photoproducts is affected to the same degree. However, since the amount of [6-41 photoproduct is only about 7%-15% of the number of cyclobutane dimers [40, 411 and its lethal effect is no greater than that of the latter [42], we believe that the presence (or absence) of this photoproduct in the
335
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profiles of pulse-labelled DNA from UV-irradiated, cytidine-pretreated Fig. 4. Sedimentation recF (A) and UV-irradiated, untreated recF uvrB (B) cells with (-) or without (. . . .) endonuclease treatment. Cells were cultured in complete medium up to a density of about 1 X IO* ml-‘, filtered, washed and resuspended in minimal medium. recF cells were subsequently cultured in the presence of 50 pg ml-’ of @dine for 90 min, filtered, washed and, after resuspending in minimal medium, irradiated with a dose of 20 J m-‘. After 110 mm cultivation in complete medium they were pulse labelled with 3H-thymidine for 10 mm. Untreated recF uvrB cells were irradiated with a dose of 10 J mm2 and, after 110 mm incubation, were pulse labellcd as above. After turning into spheroplasts, samples of each culture were divided into two aliquots, one of which was treated with endonuclease, and both were analysed in alkaline sucrose gradients. Sedimentation is from right to left.
DNA of cytidine-treated cells will not substantially influence our results. Our data suggest that the non-excisional uvr-dependent repair pathway described for wild-type cells operates in E. coli recF 143 cells, which means that the putative pathway is reck independent. The molecular mechanism of this recA &zA uvrB-dependent pathway is unknown. It has been speculated that it may be mediated by a ‘copy choice bypass’ of a lesion, where information is taken from a daughter strand of a sister duplex instead of the parental strand containing the lesion [ 191. Such an error-free bypass, mediated by the formation of a recA protein three-stranded structure and involving a breaking and rejoining step, has also been considered by other workers [43, 441.
References 1 A. Sancar and W. D. Rupp, A novel DNA repair enzyme: uvrABC excision nuclcase of Escherichia coli cuts a DNA strand on both sides of the damaged region, Cell, 33 (1983) 249-260. 2 A. T. Yeung, B. K. Jones, M. Capano and T. Chu, The repair of psoralen monoadducts by the uvrABC endonuclease, Nucleic Acids Res., 15 (1987) 4857-4971.
336 3 A. Sancar, K. A. Franklin, G. B. Sancar and M. S. Tang, Repair of psoralen and acetylamlnoiluorene DNA adducts by ABC excinuclease, J. Mol. Bid., 184 (1985) 725-734. 4 E. Seeberg, A. L. Steinum, M. Nordenskiold, S. Soderthall and B. Jcnstrom, Strand-break formation in DNA modified by benzo(a)pyrene diolepoxide. Quantitative cleavage by Escherichia coli uvrABC endonuclcase, Mutat. Res., 112 (1983) 139-145. 5 M. Fogliano and P. F. Shendel, Evidence for the inducibility of the uvrB operon, Nature, 289 (1981) 196-198. 6 C. J. Kenyon and G. C. Walker, Expression of the Escherichia coli uvrA gene is inducible, Nature, 289 (1981) 808-810. 7 G. B. Sancar, A. Sancar, J. W. Little and W. D. Rupp, The uvrB gene of Escherichia coli has both led-repressed and led-independent promotors, Cell, 28 (1982) 523-530. 8 A. Sancar, G. B. Sancar, W. D. Rupp, J. W. Little and D. W. Mount, LexA protein inhibits transcription of the Escherichia coli uvrA gene in vitro, Nature, 298 (1983) 96-98. 9 E. A. van den Berg, R. H. Geerse, J. Memelink, R. A. L. Bovenberg, F. A. Magnet and P. van de Putte, Analysis of regulatory sequences upstream of the Escherichia coli uvrB gene; involvement of the Dna.A protein, Nucleic Acids Res., 13 (1985) 1829-1840. 10 R. S. Fuller, B. E. Funnel and A. Komberg, The DnuA protein complex with the Eschenkhia coli chromosomal replication origin (orlC) and other DNA sites, Cell, 38 (1984) 889-900. 11 H. Shlzuya and D. Dykhuizen, Conditional lethality of deletions which include uvrB in stralns of Escherichia coli lacking deoxyribonucleic acid polymerasc I, J. Bacteriol., 112
(1972) 676-681. 12 M. Morimyo and Y. Shimazu, Evidence that the gene uvrB is indispensable for polymerase I deficient strain of Escherichia coli K-12, Mol. Gen. Genet., 147 (1976) 243-250. 13 T. Horiuchi and T. Nagata, Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymcrase I deficiency, Mol. Gen. Genet., I23 (1973) 89-110. 14 G. B. Smirnov, E. V. Fllkova, A. G. Skavronskaya, A. S. Saenko and B. I. Slnzlnis, Loss and restoration of viability of Escherichia coli due to combination of mutations affecting DNA polymerase I and repair activities, Mol. Gen. Genet., 121 (1973) 139-150. 15 E. C. Frledbcrg, DNA Repair, Freeman, New York, 1985, p. 234. 16 E. C. Friedbcrg, The molecular biology of nucleotide excision repair of DNA: recent progress, in A. Collins, R. T. Johnson and J. M. Boyle (cds.), Molecular biology and DNA repair, J. Cell Sci., SuppL. 6 (1987) l-23. 17 A. K. Ganesan and P. C. Seawell, The effect of ZexA and recF mutations on postreplication repair and DNA synthesis ln Escherichia coli K-12, Mol. Gen. Genet., 141 (1975) 189-206. 18 R. H. Rothman and A. J. Clark, The dependence of postrcplication repair on uvrB in a recF mutant of Escherichia coli K-12, Mol. Gen. Genet., 155 (1977) 279-286. 19 M. Sedliakova, J. Brozmanova, F. Mdek and K. Kleibl, Evidence that dlmers remaining in preinduced Escherkhiu coli B/r Her+ become insensitive after DNA replication to the extract from Micrococcus Luteus, Biophys. J., 36 (1981) 429-441. 20 J. Brozmanova, L. M&kova and M. Sedliakova, Inhibition of thymlne dimer excision after preirradiation inhibition of DNA synthesis by cytidlnc, Stud. Biophys., 36/37 (1973) 245-252. 21 M. Sedliakova, J. Brozmanova, F. M&ek and V. Slezarikova, Interaction of restoration processes lnUVirradiatedEscherichiacolicells,Photochem. Photobiol., 25 (1977) 259-264. 22 M. Sedliakova, V. Slezarikova, F. M&ek and J. Brozmanova, W-inducible repair: influence on survival, dlmer excision, DNA replication and breakdown in Escherichia coli B/r Her’ cells, Mol. Gen. Genet., 160 (1978) 81-87. 23 Z. I. Horll and A. J. Clark, Genetic analysis of the recF pathway of genetic recombination ln Escherichia coli K-12: isolation and characterization of mutants, J. Mol. BioL., 80 (1973) 327-344. 24 S. M. Picksley, R. G. Lloyd and C. Buckman, Genetic analysis and regulation of inducible recombination in Escherichia coli K-12, Cold Spring Harbor Symp. Quant. Biol., 49
(1984) 469-474. 25 B. Thorns and W. Wackemagel, Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant, J. Bacterial., 169 (1987) 1731-1736.
337 26 A. McPartland,
L. Green and H. Echols, Control of recA regulatory and signal genes, Cell, 20 (1980) 731-737. 27 J. H. Miller, Selection of thystrains with trimethoprim, 28 29 30 31
32 33
gene
RNA ln Escherichia
in Experiments
coli:
in Mobcular
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, pp. 218-220. V. Slezarikova, F. Ma&k and M. Sedliakova, Depression of dimer excision in ret _ UUT + mutant of Escherichia coli K-12, Stud. Biophys., 44 (1974) 1-6. S. S. Cohen and H. D. Bamer, Studies of unbalanced growth in Escherichia coli, Proc. Natl. Acad. Sci. U.S.A., 40 (1954) 885-893. 0. Maaloe and P. C. Hanawalt, Thymine deficiency and the normal DNA replication cycle. I, J. Mol. Biol., 3 (1961) 144-155. M. Sedliakova, F. MaSek, J. Brozmanova, L. Mdkova and V. Slezkikova, Depression of thymine dimer excision in various excision-proficient strains of Escherichia coli, Biochim. Biophys. Acta, 349 (1974) 442-446. L. G. Care and C. M. Berg, Chromosome replication in some strains of Escherichia coli K-12, Cold Spring Harbor Symp. Quant. Biol., 33 (1968) 559-573. D. Frelfelder, Technique for starvation of Escherichia coli of thymine, J. Bacterial., 90
(1965) 1153-l 154. 34 R. Mantsavinos and S. Zamenhof, Pathway for the biosynthesis of thymidilic acid in bacterial mutants, J. Biol. Chem, 236 (1961) 876-882. 35 W. L. Carrier and R. B. Setlow, The excision of pyrlmidine dimers, in L. Grosman and K. Moldave (eds.), Methods in Enzymology, Vol. XXI, Academic Press, New York, 1971, pp. 230-237. 36 R. McGrath and R. W. Wiiiams, Reconstruction in viva of irradiated Eschenfchia coli deoxyribonucleic acid: the rejoining of broken pieces, Nature, 212 (1966) 534-535. 37 R. J. Wilkins, Endonuclease-sensitive sites in the DNA of irradiated bacteria: a rapid and sensitive assay, Biochim. Biophys. Acta, 312 (1973) 33-37. 38 W. L. Carrier and R. B. Sctlow, Endonuclease from Micrococcus luteus which has activity toward ultraviolet-irradiated deoxyribonucleic acid. Purification and properties, J. Bacterial.,
IO2 (1970) 178-186. 39 M. H. Patrick, Near-UV photolysis of pyrimidine hetero adducts in Escherichia coli DNA, Photo&em. Photobiol., 11 (1970) 477-485. 40 M. H. Patrick, Studies on thymine-derived photoproducts in DNA. I. Formation and biological role of pyrimidlne adducts in DNA, Photo&em. Photobiol., 25 (1977) 357-372. 41 W. A. Franklin and W. A. Haseltine, Removal of UV light-induced pyrimidine-pyrlmidone [6-4] products from Escherichia coli DNA requires the uvrA, uvrB and uvrC gene products, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3821-3824. 42 M.-S. Thang, J. Hmclr, D. Mitchell, J. Ross and J. Clarkson, The relative cytotoxicity and mutagenicity of cyclobutanc pyrlmidlne dimers and 16-41 photoproducts in Escherichia coli cells, Mutat. Res., 161 (1986) 9-17. 43 H. Eschols, Mutation rate: some biological and biochemical considerations, Biochimie, 64 (1982) 571-575. 44 R. Woodgate, M. Rsjagopalan, Ch. Lu and H. Echols, Escherichia coli: puri6cation and interaction with Urn@ Sci. U.S.A., 86 (1989) 7301-7305.
UmuC mutagenesis protein of and UmuD’, Proc. Natl. Acad.
