Recombinational and Mutagenic Repair of Psoralen Interstrand Cross-links in Saccharomyces cerevisiae
2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês
10.1074/jbc.m103588200
ISSN1083-351X
AutoresRoss B. Greenberg, Marie Alberti, John E. Hearst, Mark A. Chua, Wilma A. Saffran,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoPsoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T → C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site. Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T → C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site. nucleotide excision repair double-strand breaks high pressure liquid chromatography base pair(s) DNA interstrand cross-linkers are used widely in cancer chemotherapy because of their high cytotoxicity in replicating cells (1Kohn K.W. Cancer Res. 1996; 56: 5533-5546PubMed Google Scholar). These lesions are complex, and their repair involves several different DNA repair pathways. As with other forms of chemical damage, excision repair systems incise the damaged DNA strands; however, there is no undamaged strand to act as a template, and full repair requires the participation of additional pathways. Recombinational repair pathways are involved in restoring the intact duplex structure after excision (2Cole R.S. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1064-1068Crossref PubMed Scopus (254) Google Scholar, 3Jachymczyk W.J. von Borstel R.C. Mowat M.R. Hastings P.J. Mol. Gen. Genet. 1981; 182: 196-205Crossref PubMed Scopus (158) Google Scholar, 4Magaña-Schwencke N. Henriques J.A. Chanet R. Moustacchi E. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1722-1726Crossref PubMed Scopus (193) Google Scholar, 5Sladek F.M. Munn M.M. Rupp W.D. Howard-Flanders P. J. Biol. Chem. 1989; 264: 6755-6765Abstract Full Text PDF PubMed Google Scholar, 6Li L. Peterson C.A. Lu X. Wei P. Legerski R.J. Mol. Cell. Biol. 1999; 19: 5619-5630Crossref PubMed Scopus (63) Google Scholar, 7McHugh P.J. Sones W.R. Hartley J.A. Mol. Cell. Biol. 2000; 20: 3425-3433Crossref PubMed Scopus (139) Google Scholar). Additionally, cross-links efficiently induce mutations, implicating error-prone pathways in their repair (8Averbeck D. Photochem. Photobiol. 1989; 50: 859-882Crossref PubMed Scopus (212) Google Scholar, 9Wang X. Peterson C.A. Zheng H. Nairn R.S. Legerski R.J. Li L. Mol. Cell. Biol. 2001; 21: 713-720Crossref PubMed Scopus (125) Google Scholar). Psoralens are photoreactive DNA cross-linking agents that react with pyrimidine bases on opposite DNA strands in the presence of near ultraviolet light; 5′-TpA-3′ sequences are preferred cross-linking sites (10Cimino G.D. Gamper H.B. Isaacs S.T. Hearst J.E. Annu. Rev. Biochem. 1985; 54: 1151-1194Crossref PubMed Scopus (656) Google Scholar, 11Tessman J.W. Isaacs S.I. Hearst J.E. Biochemistry. 1985; 24: 1669-1676Crossref PubMed Scopus (139) Google Scholar). There are two photoreactive positions in the psoralen molecule, the 4′,5′ furan and the 3,4 pyrone double bonds, which can undergo sequential photoreactions to form cross-links. The major products of the first photoreaction step are furan-side monoadducts; these can undergo a second photoreaction at the pyrone side to generate interstrand cross-links. Psoralen plus ultraviolet A (PUVA) therapy is used to treat the skin disorders psoriasis and vitiligo; although effective, this treatment has been found to induce nonmelanoma skin cancers in a dose-dependent manner (12Stern R.S. Lunder E.J. Arch. Dermatol. 1998; 134: 1582-1585Crossref PubMed Scopus (207) Google Scholar). Both nucleotide excision repair (NER)1 and recombinational repair pathways participate in the error-free repair of psoralen cross-links in Escherichia coli (2Cole R.S. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1064-1068Crossref PubMed Scopus (254) Google Scholar, 5Sladek F.M. Munn M.M. Rupp W.D. Howard-Flanders P. J. Biol. Chem. 1989; 264: 6755-6765Abstract Full Text PDF PubMed Google Scholar, 13Cole R.S. Levitan D. Sinden R. J. Mol. Biol. 1976; 103: 39-59Crossref PubMed Scopus (151) Google Scholar, 14Cheng S. Van Houten B. Gamper H.B. Sancar A. Hearst J.E. J. Biol. Chem. 1988; 263: 15110-15117Abstract Full Text PDF PubMed Google Scholar, 15Cheng S. Sancar A. Hearst J.E. Nucleic Acids Res. 1991; 19: 657-663Crossref PubMed Scopus (59) Google Scholar). The Uvr (A)BC complex makes single-strand incisions on the 5′ and 3′ sides of the cross-link. This generates an intermediate with a gap opposite to a lesion consisting of the excised oligonucleotide fragment cross-linked, through psoralen, to the uncut strand. Repair of the gap is accomplished by RecA-mediated recombination with an undamaged homologous copy of the affected DNA sequence. This yields an intact strand opposite the lesion, which can then be removed by a second round of NER. Psoralen cross-link repair in eukaryotes is not as well understood but is likely to differ from prokaryotes. In the yeast Saccharomyces cerevisiae, NER of psoralen monoadducts generates short-lived single-strand breaks that are efficiently rejoined. However, psoralen cross-links induce the formation of double-strand breaks (DSBs) in chromosomal DNA; these breaks are long-lived, and rejoining depends on homologous recombination (3Jachymczyk W.J. von Borstel R.C. Mowat M.R. Hastings P.J. Mol. Gen. Genet. 1981; 182: 196-205Crossref PubMed Scopus (158) Google Scholar, 4Magaña-Schwencke N. Henriques J.A. Chanet R. Moustacchi E. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1722-1726Crossref PubMed Scopus (193) Google Scholar, 16Miller R.D. Prakash L. Prakash S. Mol. Cell. Biol. 1982; 2: 939-948Crossref PubMed Scopus (91) Google Scholar). Production of the DSBs depends on the NER genes RAD2 and RAD3. Nitrogen mustard cross-links also induce DSBs in yeast (7McHugh P.J. Sones W.R. Hartley J.A. Mol. Cell. Biol. 2000; 20: 3425-3433Crossref PubMed Scopus (139) Google Scholar) and mammalian (17De Silva I.U. McHugh P.J. Clingen P.H. Hartley J.A. Mol. Cell. Biol. 2000; 20: 7980-7990Crossref PubMed Scopus (386) Google Scholar) cells; however, in contrast to psoralen cross-links, the formation of nitrogen mustard-induced DSBs does not depend on NER. Rejoining of psoralen cross-link-induced DSBs and regeneration of intact chromosomal DNA requires the recombinational repair genesRAD51 and RAD52 (18Averbeck D. Averbeck S. Photochem. Photobiol. 1998; 68: 289-295Crossref PubMed Scopus (28) Google Scholar). These observations suggest that error-free repair of psoralen cross-links in yeast can be carried out by a two-phase process: 1) NER acts on an interstrand cross-link to produce a DSB at the lesion site, and 2) the DSB is repaired by homologous recombination. This model predicts that, because the recombinogenic repair intermediate of a psoralen interstrand cross-link is a DSB, both psoralen cross-links and DSBs will induce similar levels and patterns of recombination. Psoralen monoadducts, which do not generate DSBs, should not be recombinogenic. We have tested these predictions by comparing the repair of plasmid molecules carrying a single site-specifically placed psoralen monoadduct, psoralen cross-link, or DSB at the same position. Induced recombination, measured as both gene conversion and crossing over, between the damaged plasmid and homologous chromosomal sequences was similar for both forms of double-strand damage. Psoralen monoadducts did not induce recombination. Psoralen cross-links, but not DSBs, also enter an alternate error-prone pathway that produces mutations at the damage site. The phis3 plasmids are yeast shuttle plasmids, derived from the phagemid vector pIBI25, carrying the yeastTRP1 and HIS3 genes. The TRP1-ARS1 EcoRI fragment from YRP12 (19Stinchcomb D.T. Thomas M. Kelly J. Selker E. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4559-4563Crossref PubMed Scopus (249) Google Scholar) was inserted into theEcoRI site of the polylinker sequence of pIBI25, and a modified his3 gene carrying an 8-bp XbaI linker insertion mutation was subcloned into the BamHI polylinker site. The plasmids phis3–75X, phis3–207X, phis3–304X, and phis3–622X have XbaI linker insertions at positions 75, 207, 304, and 622 of the HIS3 gene, respectively. To construct the his3X alleles carrying the XbaI site insertions, the XbaI site within the polylinker region of pUC18-HIS3 was first removed by digesting withXbaI, filling in the cohesive ends with Klenow fragment, and recircularizing the plasmid. XbaI linker insertions at positions 75, 207, 304, and 622 were carried out by complete or partial digestion with AvaII, MscI, HindIII, or KpnI, respectively, followed by the addition ofXbaI linkers and recircularization. XbaI linker insertion sites were confirmed by restriction mapping and DNA sequencing. Yeast strains are derivatives of W303,MATα leu2–3,112 trp1–1 ade2–1 ura3–1 can1–100 his3–11,15 rad5–535. The his3–11,15 allele was replaced with a his3X allele by two-step gene replacement (20Rothstein R.J. Methods Enzymol. 1981; 194: 281-301Crossref Scopus (1102) Google Scholar). WS101, WS102, WS103, and WS104 carry the his3 alleleshis3-75X, his3-207X, his3-304X, andhis3-622X, respectively. RY1, RY2, RY3, and RY4 areRAD5 derivatives of WS101, WS102, WS103, and WS104, respectively. The 14-bp oligonucleotide 5′-CAGGCCGTACGCAG-3′ was used for the preparation of uniquely psoralen-adducted plasmid DNA molecules. This oligonucleotide spans positions 411–424 within the coding strand of theHIS3 gene; the sequence 5′-CGTACG-3′ is a BsiWI site and contains the preferred psoralen target sequence, 5′-TA-3′. The 14-mer was annealed to the complementary 8-mer 5′-GCGTACGG-3′ and photoreacted at 366 nm with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen. Cross-linked oligonucleotides were purified by reverse-phase HPLC. The psoralen cross-links were photoreversed by irradiation at 254 nm, and 14-mers with furan-side monoadducts were resolved from 8-mers and unmodified or pyrone-side monoadducted 14-mers by reverse-phase HPLC (21Sastry S.S. Spielmann H.P. Dwyer T.J. Wemmer D.E. Hearst J.E. J. Photochem. Photobiol. B. 1992; 14: 65-79Crossref PubMed Scopus (11) Google Scholar, 22Spielmann H.P. Sastry S.S. Hearst J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4514-4518Crossref PubMed Scopus (20) Google Scholar). Plasmids with site-specifically placed psoralen monoadducts or cross-links were prepared by the extension of psoralen-modified primers annealed to single-stranded circular DNA (23Svoboda D.L. Taylor J.-S. Hearst J.E. Sancar A. J. Biol. Chem. 1993; 268: 1931-1936Abstract Full Text PDF PubMed Google Scholar). Single-stranded phagemid DNA was isolated according to Sambrook et al. (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar). Modified oligonucleotide primers were phosphorylated by treatment with T4 polynucleotide kinase and annealed to single-stranded template DNA in several 500-µl annealing reactions, each containing 5 pmol of template DNA and 160 pmol of kinased primer, in 20 mmTris-HCl, pH 7.4, 2 mm MgCl2, and 50 mm NaCl. Interstrand psoralen cross-links were formed by irradiating the annealed preparations on ice in drops of 500 µl at 365 nm for 45 min with a Schleicher and Schüell RAD-FREE long wave UV lamp with a BLE-760B Spectronics Corp. bulb. The total dose was 130 J·m−2. The irradiated samples were pooled, and noncross-linked primers were removed by ultrafiltration through Centricon-30 filters in 0.1× TEN7.4 (1 mmTris-HCl, 1 mm EDTA, pH 8.0) followed by the addition of urea to 8 m and column chromatography on Sepharose 4B in 0.1× TEN7.4. Double-stranded circular DNA was formed by primer extension of 5 pmol of primer template complex in a 650-µl reaction volume with 50 units of T4 DNA polymerase and 200 units of T4 DNA ligase in 25 mm Tris, pH 7.5, 5 mm MgCl2, 40 mm NaCl, 4 mm dithiothreitol, 7.7% glycerol, 1.5 mm ATP, and 0.8 mm each dATP, dCTP, dGTP, and dTTP for 5 min at room temperature followed by 90 min at 37 °C. Closed circular plasmid DNA molecules were prepared by centrifugation through CsCl gradients with 0.4 mg/ml ethidium bromide. The preparation of monoadduct-containing plasmid molecules was similar, but the irradiation, urea treatment, and Sepharose chromatography steps were omitted, and the annealing step was followed immediately by primer extension. Unincorporated primers remaining after the CsCl gradient were removed by urea treatment. The position of 4′-hydroxymethyl-4,5′,8-trimethylpsoralen modification within the plasmid was confirmed by BsiWI digestion. Cross-links completely inhibited and monoadducts partially inhibitedBsiWI cleavage at the target site (Fig. 1A). The presence of interstrand cross-links was verified by testing for the ability of cross-linked DNA to rapidly renature after denaturation (Fig. 1 B). Plasmid DNA (0.2 µg) was digested with BamHI, which produces a 1.8-kilobase fragment containing the HIS3 gene. The digested DNA was desalted on Sepharose spin columns to remove Mg2+, which interferes with denaturation. The samples were divided in half; one portion was denatured by incubation for 10 min with 0.2 nNaOH. Both portions were analyzed by electrophoresis on nondenaturing agarose gels in Tris acetate-EDTA buffer. The 1.8-kilobase fragment ran as double-stranded DNA, indicating that the HIS3 gene was cross-linked, whereas the larger fragment was fully denatured and ran as single strands. The plasmid preparations with furan-side monoadducts were completely denatured by this treatment. However, a 15-min irradiation of the BamHI-digested plasmid converted the 1.8-kilobase HIS3 fragment to the rapidly renaturing form, confirming that these preparations contained cross-linkable monoadducts within HIS3. A control preparation synthesized with an unmodified 14-mer primer was tested similarly for the presence of cross-links or cross-linkable adducts at the target site.BsiWI cut this preparation completely, and theBamHI fragments were fully denatured. Yeast cells were transformed with unmodified or damaged plasmid DNA as described previously (25Saffran W.A. Smith E.D. Chan S.-K. Nucleic Acids Res. 1991; 19: 5681-5687Crossref PubMed Scopus (17) Google Scholar) using 0.1 µg of plasmid and 5 µg of single-stranded carrier DNA per sample. Transformed cells were selected on tryptophan omission medium, and colonies were scored after 4 days. Repair efficiency was calculated as the ratio of Trp+transformants with damaged plasmid to Trp+ transformants with undamaged plasmid. Trp+ colonies were replica-plated to histidine omission medium to determine the histidine phenotype. Plasmid integration was measured by determining the stability of the Trp+ and His+ phenotypes; Trp+colonies were serially replicated to three yeast extract-peptone-dextrose plates to dilute out extrachromosomally replicating plasmid, then replicated back to synthetic dextrose-Trp and synthetic dextrose-His plates (26Han E.-K. Saffran W.A. Mol. Gen. Genet. 1992; 236: 8-16Crossref PubMed Scopus (4) Google Scholar). Plasmids were transferred from yeast toE. coli by a modification of the method of Strathern and Higgins (27Strathern J.N. Higgins D.R. Methods Enzymol. 1991; 194: 319-329Crossref PubMed Scopus (124) Google Scholar). Yeast cultures were grown 2 days in 2 ml of yeast extract-peptone-dextrose medium at 30 °C. The cells were harvested and resuspended in 0.1 ml of lysis buffer (2.5 mLiCl, 50 mm Tris-HCl, pH 8.0, 20 mm EDTA, and 4% Triton X-100). An equal volume of phenol/chloroform/isoamyl alcohol plus one-third volume of acid-washed glass beads (0.45–0.50 mm) were added, and the mixture was vortexed vigorously for 10 min. The mixture was incubated at 65 °C for 5 min and then centrifuged; the aqueous phase was recovered and re-extracted with phenol/chloroform/isoamyl alcohol. The preparation was purified over 0.5 ml of Wizard miniprep resin (Promega) according to manufacturer directions using column wash buffer consisting of 55% ethanol, 200 mm NaCl, 20 mm Tris-HCl, and 5 mm EDTA, pH 7.5. The DNA was eluted from the resin in 50 µl of TE buffer (10 mmTris-HCl, 1 mm EDTA, pH 8.0). Competent cells prepared by the method of Inoue et al. (28Inoue H. Nojima H. Okayama H. Gene (Amst.). 1990; 96: 23-28Crossref PubMed Scopus (1569) Google Scholar) were transformed with 10 µl of the DNA preparation, and minipreps were isolated from bacterial cells by alkaline lysis (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar). All plasmid analyses were done on duplicate colonies from the E. coli transformation step. Plasmid DNA was digested with XbaI to monitor gene conversion between plasmid and chromosome his3X alleles. All the plasmids used contain an invariant 3046-bp XbaI fragment. The remaining 3061-bp fragment is intact in plasmids with a wild-type HIS3 gene and is cleaved into two fragments of 1228 and 1833 bp in phis3–75X, 1096 and 1965 bp in phis3–207X, 999 and 2062 bp in phis3–304X, and 678 and 2383 bp in phis3–622X. Mutations at the damage target site were detected by BsiWI digestion. The plasmids that were not cut by BsiWI were subjected to DNA sequencing. Southern analysis of samples with integrated plasmids was performed as described previously (29Saffran W.A. Cantor C.R. Smith E.D. Magdi M. Mutat. Res. 1992; 274: 1-9Crossref PubMed Scopus (15) Google Scholar). Genomic DNA was digested with EcoRI to distinguish single and multiple plasmid integrations and with XbaI to characterize gene conversion. The his3-75X, his3-207X,his3-304X, and his3-622X alleles generatedXbaI digestion fragments of 1804, 1672, 1575, and 1257 bp, respectively, from the downstream flanking chromosomal XbaI site; in addition, the integrated plasmids produced the sameXbaI fragments as the extrachromosomal plasmids as described above. A combination of genetic and physical analysis was used to construct gene conversion spectra. Colonies were determined to carry extrachromosomal or integrated plasmids by genetic characterization of Trp+ stability, and the two categories were analyzed separately. Direct assignments to some classes were made for colonies with unambiguous phenotypes,i.e. His+ or His± extrachromosomal plasmids. Physical analysis by Southern hybridization or plasmid rescue was carried out on samples with ambiguous phenotypes, i.e.integrated plasmids and His− extrachromosomal plasmids. The frequency of each conversion class, determined by physical analysis, was applied to the total fraction of colonies with a given phenotype to calculate the final conversion spectrum. Total genomic DNA was isolated according to Sherman et al. (30Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1986Google Scholar). The primers used to amplify the HIS3 gene were 5′-TCCACCTAGCGGATGACTCT-3′ and 5′-CACTTGCCACCTATCACCAC-3′. Polymerase chain reaction was carried out for 25 cycles of 1 min at 94 °C and 2 min at 60 °C in 2.5 mm MgCl2, 0.25 µm of each primer, and 0.2 mm each dATP, dCTP, dGTP, and dTTP. The amplified DNA was digested with BsiWI to detect targeted mutations. Plasmid DNA molecules carrying single site-specifically placed lesions were prepared in vitro and introduced into yeast cells forin vivo repair. The lesion site was the uniqueBsiWI restriction site within the HIS3 gene at position 416. This site was chosen because it contains a preferred 5′-TA-3′ sequence for psoralen photoaddition. DNA repair substrates with targeted psoralen adducts were prepared by annealing 14-base oligonucleotides modified with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen furan-side monoadducts to single-stranded circular DNA. Irradiation with long wave UV light converted the monoadducts to cross-links. Primer extension of the cross-linked primer produced double-stranded circular plasmid molecules with cross-links at the BsiWI site. The plasmids with targeted psoralen monoadducts were formed similarly except the UV irradiation step was omitted. DSBs were produced by BsiWI digestion. The modified plasmid molecules were transfected into yeast cells, and the transformation efficiency relative to that of undamaged plasmid was used as a measure of repair. The plasmids contain, in addition to thehis3 target gene, a copy of TRP1; we followed transformation as the appearance of Trp+ colonies. The repair efficiencies for DSBs, psoralen monoadducts, and psoralen cross-links are presented in Table I. Single psoralen cross-links were repaired less efficiently, by a factor of 2–3, than either single psoralen monoadducts or DSBs placed at the same site, confirming that these complex double-strand lesions are more difficult for yeast cells to repair than single-strand lesions or clean breaks.Table IRepair of damaged plasmid DNA in yeast cellsDamageRelative transformation efficiency1-aThe number of Trp+transformants was normalized to the number of Trp+ colonies resulting from transformation by undamaged plasmid. The reported values are the means and standard deviations of 12 determinations. The repair efficiency of psoralen cross-links is significantly lower than that of double-strand breaks (p < 0.01) and psoralen monoadducts (p < 0.001) by t test analysis.None(1.00)Double-strand break0.41 ± 0.13Psoralen monoadduct0.61 ± 0.12Psoralen cross-link0.17 ± 0.081-a The number of Trp+transformants was normalized to the number of Trp+ colonies resulting from transformation by undamaged plasmid. The reported values are the means and standard deviations of 12 determinations. The repair efficiency of psoralen cross-links is significantly lower than that of double-strand breaks (p < 0.01) and psoralen monoadducts (p < 0.001) by t test analysis. Open table in a new tab The major pathway for repair of double-strand DNA breaks in yeast is through homologous recombination (31Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). Psoralen interstrand cross-links are processed to DSBs by the nucleotide excision repair pathway (3Jachymczyk W.J. von Borstel R.C. Mowat M.R. Hastings P.J. Mol. Gen. Genet. 1981; 182: 196-205Crossref PubMed Scopus (158) Google Scholar, 4Magaña-Schwencke N. Henriques J.A. Chanet R. Moustacchi E. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1722-1726Crossref PubMed Scopus (193) Google Scholar) and also stimulate homologous recombination (25Saffran W.A. Smith E.D. Chan S.-K. Nucleic Acids Res. 1991; 19: 5681-5687Crossref PubMed Scopus (17) Google Scholar, 32Meira L. Henriques J.A.P. Magaña-Schwencke N. Nucleic Acids Res. 1995; 23: 1614-1620Crossref PubMed Scopus (3) Google Scholar). Psoralen monoadducts, in contrast, are processed by nucleotide excision repair to form single-strand breaks and are less efficient in stimulating recombination (33Saffran W.A. Greenberg R.B. Thaler-Scheer M.S. Jones M.M. Nucleic Acids Res. 1994; 22: 2823-2829Crossref PubMed Scopus (30) Google Scholar). We compared the abilities of double-strand breaks and both forms of psoralen photodamage to stimulate crossing over and gene conversion (31Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). Gene conversion, defined as the transfer of genetic information from a donor DNA molecule to a recipient, is a consequence of recombinational repair. Repair replication, initiated from an invading strand of the damaged DNA molecule on the undamaged molecule, can form a segment of asymmetric heteroduplex DNA. Branch migration of the Holliday junction joining the two molecules may further extend the region of heteroduplex DNA symmetrically on both damaged and undamaged DNA. Mismatch repair processes mismatches within the heteroduplex DNA to generate conversions. Genetic exchanges between damaged plasmid and undamaged chromosomal DNA molecules were followed by analyzing the retention of markers placed at distances of 100–300 bp from the damage site within theHIS3 gene. In all experiments the damage consisting of a double-strand break, psoralen monoadduct, or psoralen cross-link,was located at the BsiWI site at position 416 of the plasmidHIS3 gene. Genetic markers consisting of XbaI linker insertions were placed at positions 75, 207, 304, or 622, producing the his3 alleles his3-75X,his3-207X, his3-304X, and his3-622X, respectively. Each his3 allele contained only oneXbaI linker insertion mutation; we thus were able to detect recombination events between pairs of his3 alleles that led to changes in the His phenotype. We were able to measure gene conversion at two positions in each experiment, corresponding to the sites of the XbaI markers in the plasmid and chromosome HIS3 alleles. Some of the conversion events gave rise to phenotypic changes that could be detected by genetic analysis. The plasmid repair substrate carrying a marker at position 622 of the HIS3 gene was most informative in this respect (Fig. 2). The coding region of the HIS3 gene is only 663 nucleotides in length, and the insertion at position 622 places the mutation near the C terminus of the gene product. Cells with the multicopy plasmid phis3–622X show a slow growth phenotype in histidine omission medium, indicating that there is residual enzymatic activity in these mutants; we denote this phenotype by His±. Cells with the corresponding chromosomal his3-622X allele in single copy do not grow in the absence of histidine, suggesting that overexpression of the mutant his3-622X gene is necessary for the residual growth that we observed. Fig. 2 illustrates the possible outcomes of gene conversion in extrachromosomal phis3–622X. Conversion tracts are assumed to initiate at the damage site at position 416 and to extend continuously in one or both directions from this point, and the damaged plasmid is assumed to be the recipient (chromosome-to-plasmid conversion). No conversion, or short tracts that do not reach either marker, will produce an unchanged phis3–622X with a His± phenotype (Fig. 2 A). Unidirectional conversion in the upstream direction transfers the chromosome marker to the plasmid, generating a double mutant plasmid with a His− phenotype (Fig. 2 B). Unidirectional conversion in the downstream direction replaces the plasmid marker with the corresponding wild-type sequence, generating HIS3 with a His+ phenotype (Fig. 2 C). Finally, bidirectional gene conversion replaces the plasmid his3-622X allele with the chromosomal allele, which has a His− phenotype (Fig.2 D). This outcome cannot be distinguished from the product of upstream conversion by its phenotype, because both are His−. The observation of phenotypic changes depends on the length of the conversion tract; the conversion frequency is expected to be higher close to the damage site if most tracts are short. For extrachromosomal plasmids, His− colonies can be produced by upstream or bidirectional conversion tracts that transfer the chromosomal marker to the plasmid (Fig. 2, B and D); markers closer to the initiation site should be transferred at higher frequencies than more distant markers. The strain with a marker at position 304, about 100 bp away from the damage site, had a slightly higher frequency of cross-link-induced His− colonies than the strains with markers at positions 75 or 207 (Fig. 3), but the difference was not significant (χ2 = 3.3,p
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