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Originally published In Press as doi:10.1074/jbc.M402323200 on June 22, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36462-36469, August 27, 2004
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DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways*

Wilma A. Saffran{ddagger}§, Shaila Ahmed{ddagger}, Sherly Bellevue{ddagger}, Gillian Pereira{ddagger}, Teleka Patrick{ddagger}, Wendy Sanchez{ddagger}, Sandra Thomas{ddagger}, Marie Alberti¶, and John E. Hearst¶

From the {ddagger}Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, New York 11367 and Department of Chemistry, University of California and Lawrence Berkeley National Laboratory, Berkeley, California 94720

Received for publication, March 1, 2004 , and in revised form, June 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent errorprone pathways, one NER-dependent and one NER-independent.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA interstrand cross-links are complex lesions, highly toxic to cells, and difficult to repair because of the involvement of both strands of the DNA duplex. A single unrepaired interstrand cross-link is sufficient to kill a cell, and multiple DNA repair pathways may be required to complete cross-link removal (1–7).

The yeast Saccharomyces cerevisiae has three major DNA repair epistasis groups, all of which are involved in cross-link repair; they are nucleotide excision repair, recombinational repair, and post-replication repair (8). Nucleotide excision repair (NER)1 is a general system that recognizes bulky and helix-distorting lesions (9, 10). Endonucleases nick the affected DNA strand on both the 5' and 3' sides of the lesion; after removal of the damaged oligonucleotide, the resulting singlestrand gap is filled in by polymerase activity using the undamaged strand as a template. This mechanism produces error-free repair of single-strand damage. Recombinational repair is the major pathway for repair of double-strand damage, such as double-strand breaks (DSBs) or gaps in yeast (11, 12). In homologous recombination (HR) the broken DNA molecule is repaired, and missing genetic information is restored through interactions with intact homologous sequences. This pathway is also non-mutagenic but can result in DNA rearrangements. Post-replication repair (PRR) acts on damage in the context of DNA replication, allowing for bypass of replication fork-blocking lesions; this pathway, rather than removing lesions, permits replicating cells to tolerate the damage (13, 14). Lesion bypass may be achieved by error-prone mechanisms, such as trans-lesion synthesis by specialized DNA repair polymerases, or by error-free mechanisms, such as template switching.

Psoralen undergoes photoreaction with DNA to form monoadducts and interstrand cross-links (15, 16). Psoralen photoreaction of replicating yeast cells induces NER-dependent DNA breaks; monoadducts induce single-strand nicks, whereas interstrand cross-links induce DSBs (2, 5, 17–20). Rejoining of the cross-link-induced DSBs depends on the HR genes RAD51 and RAD52 (2, 5, 21–23). These observations suggest the following two-step model for psoralen cross-link repair; 1) NER acts on cross-links to produce DSBs, and 2) the DSBs are repaired by HR. Evidence in support of this pathway includes the sensitivities of NER- and recombination-deficient yeast strains to psoralen photoreaction and the induction of homologous recombination by cross-links (24–28). However, an additional pathway for cross-link repair exists, as indicated by the sensitivity of PRR-deficient yeast cells and cross-link-induced mutagenesis (26–29). Interstrand cross-links, therefore, appear to be repaired by alternate pathways involving recombination or mutation.

In a previous study of psoralen photodamage repair in repair-proficient yeast cells (30) we found that interstrand cross-links placed at a specific site within plasmid molecules induced homologous recombination in the majority of repair events and mutations targeted to the damage site in a smaller fraction of the repaired plasmids. There was no overlap between these endpoints, suggesting that the recombinational and errorprone pathways are independent, alternate mechanisms for interstrand cross-link repair. A model for psoralen cross-link repair is shown in Fig. 1. In the current study we have characterized the repair of these plasmid molecules in yeast strains with deficiencies in each of the three major DNA repair epistasis groups. We find that blocking either NER or HR channels cross-links into mutagenic repair, whereas blocking PRR channels cross-links into recombinational repair.



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  		FIG. 1.
Involvement of the major DNA repair epistasis groups in psoralen cross-link repair. Psoralen cross-link processing to DSBs requires nucleotide excision repair; the DSBs are repaired by homologous recombination. An alternative pathway, depending on post-replication repair, produces mutations.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Plasmids—Yeast strains were constructed from W1588–4A, MAT{alpha} his3-11,15 ura3-1 leu2-3,112 trp1-1 ade2-1 can1-100, which is a RAD5 derivative of W303 (31). The his3-11,15 allele was replaced by his3-207X; this allele is an XbaI linker insertion at position 207 of the HIS3 gene. Isogenic repair-deficient strains carry one of the following alleles: rad1::LEU2, rad3–2, rad4::LEU2, rad5::URA3, rad6::URA3, rad10::LEU2, rad18::URA3, rad51::LEU2, rad52::LEU2, rad54::LEU2, rad55::LEU2, or rad57::LEU2. The plasmid phis3-622X is derived from the phagemid vector pIBI25; it carries a TRP1-ARS1 fragment as well as the his3-622X allele, which has an XbaI insertion at position 622. Plasmid phis3-207X is similar to phis3-622X but with an XbaI insertion at position 207.

Preparation of Plasmids with Site-specific Damage—Plasmids with DSBs were prepared by digestion with BsiWI. Plasmids with psoralen monoadducts or cross-links were prepared as previously described (30). Briefly, a 14-base oligonucleotide primer, 5'-CAGGCCGTACGCAG-3', was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen to produce an adduct at the unique T position. For preparation of monoadducted plasmids the modified primer was annealed to the single-stranded form of the phagemid and extended with DNA polymerase to form the second strand. For generation of cross-linked plasmid the annealed preparation of primer with single-stranded circles was irradiated with near ultraviolet light to convert the monoadducts to cross-links before second-strand synthesis. Covalently closed circles were formed with DNA ligase and purified by centrifugation through CsCl gradients in the presence of ethidium bromide.

Plasmid Repair and Genetic Analysis—Yeast cells were converted to spheroplasts and transformed with modified plasmids as previously described (30) 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. Trp+ colonies were replica-plated to histidine omission medium to determine the histidine phenotype; growth after 1 day was scored as His+, and growth after 2 days was scored as His+/–. 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 tryptophan and histidine omission plates. For experiments with plasmid phis3-207X only repair and plasmid integration were measured.

Physical Analysis—Plasmid DNA was amplified by polymerase chain reaction to characterize gene conversion and mutation patterns. Primers VF1 (5'-ACACTTTATGCTTCCGGCTCG-3') and HF2 (5'-CTTAGCCATTGGCATTATCACA-3') were used to amplify the plasmid HIS3 gene only. Reactions were performed in 2.0 mM magnesium, 200 µM each dNTP, 0.25 µM each primer, and 10 ng of template DNA. Amplification was carried out for 30 cycles of 2 min at 94 °C, 2 min at 55 °C, and 2 min at 77 °C. The amplification product was digested with XbaI to characterize gene conversion. The his3-622X allele yields three fragments of 164, 682, and 729 bp with XbaI digestion. Conversion to his3-207X produces fragments of 164, 314, and 1097 bp, conversion to his3-207X,622X produces fragments of 164, 314, 415, and 682 bp, and conversion to HIS3 produces two fragments of 164 and 1411 bp. The amplification product was also digested with BsiWI to detect mutations at the damage site; phis3-622X yields fragments of 523 and 1052 bp, whereas mutants are refractory to BsiWI digestion. Integrated plasmids were characterized by Southern analysis as previously described (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental Design—Plasmid molecules were modified in vitro to produce DNA damage at a unique site. Plasmid phis3-622X is a yeast replicating plasmid carrying the TRP1 and HIS3 genes. It contains an ARS replication origin but no centromere sequence. Psoralen monoadducts or interstrand cross-links were added at the unique BsiWI site at position 418 in the HIS3 gene; the BsiWI recognition sequence, 5'-CGTACG-3', includes a preferential psoralen binding site. DSBs were produced at the same site by BsiWI digestion. Yeast cells were transformed with intact or modified plasmids, and repair was measured as the transformation efficiency of damaged plasmid relative to undamaged plasmid. Transformation was monitored as the appearance of Trp+ colonies. The repair efficiencies are shown in Table I.


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  		TABLE I
DNA damage repair

 
Colonies carrying repaired plasmids underwent genetic and physical analysis to detect mutation and recombination. Homologous recombination produces crossing-over and gene conversion. Crossing-over leading to plasmid integration was detected as stable Trp+ colonies; the extrachromosomal plasmids, lacking centromeres, are unstable under nonselective conditions. The frequencies of plasmid integration are presented in Table II. Gene conversion within HIS3 was detected by growth rate on histidine omission medium. The his3-622X allele has an 8-base pair XbaI linker insertion at position 622 of HIS3. This mutation is near the end of the coding region, and the gene product retains residual activity. Yeast cells carrying this allele on a multicopy plasmid grow slowly on histidine omission medium; this phenotype is denoted as His+/–. Conversion between the damaged plasmid and undamaged chromosomal his3 allele produces a phenotypic change to His+ or His–, as depicted in Fig. 2. Conversion in the downstream (3') direction produces His+ colonies that grow quickly on histidine omission medium, whereas conversion in the upstream (5') direction and bidirectional conversion produce His– colonies that show no growth in the absence of histidine. Mutations at the damage site generally inactivate the gene, also producing a His– phenotype. Phenotypic changes to His+ and His– are given in Table III.


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  		TABLE II
Damage-induced plasmid integration

 



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  		FIG. 2.
His phenotype dependence on the direction of gene conversion tracts in the his3-622X allele. A multicopy plasmid with the his3-622X allele grows slowly in histidine omission medium, phenotype His+/–. The damage is located at the BsiWI site, position 416. Gene conversion starting from the damage site using the chromosomal his3-207X allele as the donor generates a his3-207X,622X allele with a His– phenotype in the 5' (upstream) direction, a HIS3 allele with a His+ phenotype in the 3' (downstream) direction, and a his3-207X allele with a His– phenotype if the conversion tract is bidirectional.

 


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  		TABLE III
Damage-induced phenotypic changes

 
Colonies were further characterized by physical analysis. Integrated plasmids were studied by Southern analysis, and extrachromosomal plasmids were amplified by PCR or by transformation into bacterial cells. Gene conversion was characterized by changes in the XbaI restriction pattern to distinguish between upstream, downstream, and bidirectional conversion. Targeted mutations were detected by loss of the BsiWI cutting site. Gene conversion and mutation frequencies of cross-linked plasmids are given in Table IV.


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  		TABLE IV
Psoralen cross-link-induced gene conversion and mutation

 
Repair-proficient Cells—Plasmids carrying DSBs, psoralen monoadducts, or psoralen cross-links at the unique BsiWI site of phis3-622X were prepared in vitro and transformed into repair-proficient yeast cells for in vivo repair. Monoadducts were efficiently repaired, showing a transformation frequency of 60% relative to undamaged plasmids (Table I). DSBs were also fairly well repaired, with a relative transformation frequency of 45%. The repair efficiency of psoralen cross-links, at 20%, was lower than that of the monoadducts or DSBs. Both forms of double-strand damage induced plasmid integration, whereas psoralen monoadducts did not; DSBs induced higher levels of integration than cross-links (Table II). Double-strand damage also induced phenotypic changes to His+ and His– (Table III). His+ colonies are generated by gene conversion, but His– colonies can be produced by mutations as well as gene conversions. For DSBs, almost all the His– colonies were produced by gene conversion, but many of the cross-link-induced His– colonies were the result of mutations at the damage site rather than gene conversion (Table IV). Cross-links, thus, appear to be repaired by alternate pathways leading to either homologous recombination or mutations. We examined the repair products of DSB, psoralen monoadduct, and psoralen cross-link modification in yeast strains deficient in the NER, HR, or PRR pathways to characterize the contributions of each pathway to these recombinational and mutagenic endpoints.

Nucleotide Excision Repair-deficient Strains—Psoralen monoadducts and cross-links are both acted on by the NER pathway in yeast; monoadducts yield single-strand breaks, whereas DSBs are produced in response to psoralen cross-links. That DSBs are effective inducers of homologous recombination leads to the prediction that psoralen cross-link-induced recombination should show dependence on NER. DNA damage-induced repair, recombination, and mutation were examined in yeast strains deficient in the NER genes RAD1, RAD3, RAD4, and RAD10.

DSB repair efficiency was unaffected by loss of RAD3 or RAD4 function but was lower in rad1 and rad10 strains (Table I). Rad1 and Rad10 form a heterodimeric protein that makes the 5' side incisions during the excision stage of NER (32, 33). In addition, the Rad1·Rad10 complex acts in recombination to cleave at double strand-single strand junctions and functions in DSB-induced plasmid integration (34, 35) as we observe here (Table II). DSB-induced conversion to His+ was also reduced in these strains (Table III).

Psoralen monoadduct repair was unaffected or slightly reduced by deficiencies in the NER genes. Although psoralen monoadducts are substrates for NER, a single lesion on an extrachromosomally replicating plasmid did not greatly reduce the measured transformation level, perhaps because of replication of the undamaged strand (36). As with repair-proficient cells, monoadducts did not induce plasmid integration or conversion to His+.

NER deficiencies reduced the level of psoralen cross-link repair. Cross-link-induced plasmid integration and conversion to His+ were also dependent on NER function. Although the dependence of recombination on RAD1 and RAD10 could have been due to involvement of these genes in recombination rather than in the processing of interstrand cross-links to recombinogenic intermediates, cross-link-induced integration and conversion also depended on RAD3 and RAD4. This observation is consistent with action of the NER pathway on psoralen cross-links to produce intermediates, possibly DSBs, which induce recombination.

Physical analysis of repaired plasmids from NER-deficient cells confirmed that gene conversion was reduced in all the strains (Table IV). The overall occurrence of His+ colonies was reduced to a greater extent than His– colonies (Table III) in comparison to repair-proficient cells; this suggests that an additional repair process resulting in a His– phenotype is active in NER-deficient yeast. In addition to homologous recombination, interstrand cross-links can be repaired by the error-prone PRR pathway, resulting in mutations. We found mutations at the cross-link site in 12–20% of the plasmids repaired in NER-deficient cells in comparison with a mutation frequency of 5% in repair-proficient cells (Table IV). Sequencing of several mutated plasmids showed that all had base substitutions at the cross-link site (Table V). These results indicate that mutagenic repair plays a larger role and recombinational repair a smaller role in NER-deficient than in repair-proficient cells.


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  		TABLE V
Psoralen cross-link-induced mutations

The psoralen interstrand cross-link was formed between positions 418 and 419. The psoralen molecule formed a furan-side adduct with the thymidine at position 418, within the non-transcribed strand, and a pyrone-side adduct with the thymidine at position 419, within the transcribed strand.

 
Homologous Recombination-deficient Strains—HR is the major repair pathway for DSB repair in S. cerevisiae (11), and the induction of recombination by interstrand cross-links is thought to be due to DSB-containing intermediates in cross-link repair (1, 6, 7, 30). We examined repair, recombination, and mutation in strains deficient in the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57. DSB repair was reduced, as expected, in these strains in comparison to homologous recombination-proficient cells (Table I). DSB-induced plasmid integration and conversion to His+ were decreased in these strains, indicating that the residual repair was likely due to non-homologous end-joining, which does not produce crossingover or gene conversion (Tables II and III).

HR does not play a large role in psoralen monoadduct repair; the repair efficiency of monoadducted plasmids was not substantially decreased in these strains, and the levels of plasmid integration and conversion to His+ were low, as in the repair-proficient cells. Recombinational repair of interstrand cross-links is thought to occur by a two-step process in which NER first acts to produce DSBs, and break-rejoining mediated by HR is then accomplished by genes of the RAD52 epistasis group. Recombination-deficient yeast strains are sensitive to psoralen photoreaction; however, repair of plasmids cross-linked at a single site showed limited dependence on genes in this group. The rad55 and rad57 strains were more sensitive than the rad51, rad52, and rad54 strains, with a roughly 2-fold decrease in repair efficiency relative to repair-proficient cells (Table I). RAD55 and RAD57 are paralogs of the strand exchange protein RAD51; in mammalian cells defects in the RAD51 paralogs XRCC2 and XRCC3 confer high sensitivities to cross-linking agents (37). In keeping with the deficiencies in HR, there was little cross-link-induced plasmid integration or gene conversion to His+ in any of these strains.

As in the NER-deficient strains, although His+ levels were low, His– frequencies were not substantially decreased in recombination-deficient cells, suggesting that mutagenic repair was active. Physical analysis of repaired plasmids confirmed that little or no gene conversion was induced by psoralen cross-links, but that about 20 to 30% carried mutations at the damage site (Table IV). These levels were higher than those in repair-proficient cells and also higher than mutation frequencies in the NER-deficient strains. Sequencing of plasmids confirmed the presence of targeted mutations, all base substitutions, at the damage site (Table V). Thus, error-prone repair is a major pathway for psoralen interstrand cross-link repair in the absence of HR.

Post-replication Repair-deficient Strains—The PRR pathway allows the cell to tolerate and bypass rather than remove damage by error-prone and error-free subpathways. Although this is not a major pathway for DSB repair, DSB repair was slightly decreased relative to repair-proficient cells in rad5, rad6, and rad18 yeast (Table I). However, homologous recombination, measured as levels of plasmid integration and gene conversion, was not decreased in these strains (Tables II and III). Repair and recombination of monoadducts were also unaffected by deletion of PRR genes.

Psoralen interstrand cross-links are highly mutagenic, indicating that error-prone repair is active on these lesions. Cross-link repair in these plasmids depended on the RAD5, RAD6, and RAD18 genes, with repair levels decreased 3–5-fold relative to repair-proficient cells (Table I). Measurement of recombination showed that both crossing-over and gene conversion were increased relative to repair-proficient cells by deficiencies in PRR. Plasmid integration was about 50% higher, and conversion to His+ was stimulated by 50–100% in these strains (Tables II and III). In contrast, mutation levels were decreased, with no mutations found among the 165 samples in this group that were analyzed. All the His– colonies were produced by gene conversion, producing overall gene conversion frequencies equal to (in rad6 and rad18) or higher than (in rad5) repair-proficient yeast (Table IV). Thus, in yeast cells lacking the PRR pathway, cross-linked plasmids were repaired by HR.

The pattern of phenotypic change induced in the rad5 and rad6 yeast by psoralen cross-linking differed from that seen in the other strains; there were higher frequencies of His+ than His– colonies in these two strains, in contrast to the greater incidence of His– colonies in the repair-proficient and the other repair-deficient cells (Table III). Physical analysis confirmed that the difference was due to the direction of gene conversion. In previous studies in repair-proficient cells (30) we observed a bias in the directionality of damage-induced gene conversion within HIS3. DSBs placed in the middle of the HIS3 gene induced more conversion in the 3' (downstream) direction than in the 5' (upstream) direction. Psoralen cross-links also induced asymmetric gene conversion tracts but with the opposite bias; we observed higher levels of upstream than downstream conversion tracts. In the present study we observed similar patterns for both DSB- and cross-link-induced gene conversions in the rad5 and rad6 strains (Table VI). Both forms of double-strand damage yielded asymmetric tracts, with a bias toward downstream conversion. Loss of RAD5 or RAD6 function, therefore, affects the type as well as the level of gene conversion. In the rad18 strain, in contrast, both DSB and cross-link-induced conversions showed a bias toward upstream conversion tracts.


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  		TABLE VI
Asymmetrical gene conversion patterns

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In repair-proficient yeast cells psoralen cross-links are repaired by recombinational and mutagenic pathways. Recombinational cross-link repair is thought to involve sequential action of NER and HR, whereas mutagenic repair requires genes of the PRR pathway. We tested the model shown in Fig. 1 by separately blocking each of the major DNA repair epistasis groups and measuring the effects on repair, recombination, and mutation.

Recombinational Repair—Interstrand cross-links produced by psoralen and other agents are processed to DSBs, which are thought to be the recombinogenic intermediates in cross-link repair by homologous recombination. In yeast cells the formation of psoralen-induced DSBs depends upon NER function (2, 5, 19, 20). However, NER is dispensable for the generation of DSBs by nitrogen mustard cross-links in both yeast and mammalian cells (6, 23). Cross-links of both types induce DSBs in replicating but not stationary phase cells (2, 19, 23); in addition, psoralen cross-links must pass through S phase to produce cell cycle arrest and chromosome breakage in mammalian cells (38), and cross-link-induced DSBs are formed in S phase (39). In vitro, DSBs are induced near psoralen cross-links during replication initiating at an SV40 origin (40). These observations suggest that DSBs are not formed directly by NER or other nuclease action on cross-links. Instead, DSBs form in S phase when cross-links or perhaps cross-link repair intermediates encounter and stall a replication fork.

The mammalian NER factors act on psoralen interstrand cross-links in vitro to produce two incisions, both 5' to the cross-link, but leave the cross-link in place (41). Cross-links can be unhooked by the exonuclease activity of XPF-ERCC1 (41), the NER factor homologous to S. cerevisiae Rad1·Rad10. The importance of this factor in mammalian cross-link repair is indicated by the high sensitivity of XPF- or ERCC1-deficient cells to cross-linking agents (42). In contrast to mammalian cells, RAD1- or RAD10-deficient yeast strains are not more sensitive to psoralen photoreaction than other NER-deficient strains (29), suggesting that psoralen cross-link repair is different in this lower eukaryote.

We observed that deletion of NER genes reduced but did not eliminate psoralen cross-link-induced recombination, measured as crossing-over (plasmid integration) or as gene conversions. This indicates that, although most recombination does depend on NER, there is an additional minor NER-independent component. It is possible that, as for nitrogen mustard cross-links, DSBs are generated by an NER-independent mechanism. Measurements of psoralen-induced DSBs by pulsed field gel electrophoresis showed a larger reduction in a triple rad2 rad6 rad52 mutant strain than in the single rad2 mutant, indicating a role for other repair epistasis groups in DSB formation (19). Alternatively, DSBs may not be the only recombinogenic intermediates; for instance, in bacteria recombination is initiated by single strand gaps opposite psoralen adducts (3, 4).

Cross-link-induced recombination depended on the genes of the RAD52 epistasis group. However, although recombinational repair was blocked in HR-deficient strains, psoralen cross-links were still repaired fairly efficiently by the alternate error-prone pathway. Similarly, deficiencies in the post-replication repair genes RAD5, RAD6, and RAD18 channeled cross-links into the recombinational repair pathway, as indicated by the elevated levels of gene conversion and plasmid integration in repaired plasmids. Loss of RAD5 or RAD6 function affected the nature as well as the level of cross-link-induced recombination. In repair-proficient cells DSBs and cross-links generate gene conversion tracts with opposite directional asymmetries (30); this difference disappeared in rad5 and rad6 yeast, with cross-link-induced conversions assuming the DSB pattern. RAD5 and RAD6 may act in the formation of recombinogenic intermediates, thus influencing the outcome of recombination, or at subsequent steps in recombination.

RAD5 is a member of the error-free branch of the RAD6 PRR epistasis group (43, 44). Rad6 is a ubiquitin-conjugating enzyme required for all forms of PRR; it forms a complex with the RING finger protein Rad18, which binds single-stranded DNA (45). The Rad6-Rad18 heterodimer monoubiquitinates the DNA replication and repair factor PCNA (46). Rad5 is homologous to the SWI/SNF family of chromatin-remodeling proteins and also has a RING finger domain. It associates with the Ubc13-Mms2 ubiquitin-conjugating enzyme to attach additional ubiquitins to the monubiquitinated PCNA (46). The RAD5-UBC13-MMS2 branch of post-replication repair is thought to promote the error-free bypass of replication fork-blocking lesions through recombinational mechanisms such as replication restart or template switching (13). Rad5 may have functions in addition to those in the UBC13-MMS2 pathway, as rad5 cells are more UV-sensitive than mms2 cells (44). A requirement for RAD5 in the excision of DNA minor groove adducts has been reported, suggesting a possible role in the generation of recombinogenic intermediates (47). RAD5 is also involved in the entry of DSBs into HR rather than non-homologous end-joining (48) and similarly may affect the entry of cross-link repair intermediates into HR.

Unlike rad5 and rad6, the rad18 mutation did not affect the cross-link-induced gene conversion pattern; this strain displayed the same bias as the repair-proficient cells, with more upstream than downstream conversion. However, the RAD18 deficiency affected the DSB-induced conversions, producing the same preponderance of upstream conversions as cross-links. In contrast, RAD5 and RAD6 deletions did not affect the DSB-induced conversion pattern. RAD18 deficiency in chicken lymphocytes has been reported to decrease targeted integrations (49), indicating that this PRR gene affects some forms of homologous recombination.

Error-prone Repair—In repair-proficient yeast error-prone repair is a minor pathway for psoralen cross-links, but in strains lacking either NER or HR it is the major pathway, as indicated by the higher levels of cross-link-induced mutation than recombination. We found no cross-link-induced mutations in strains lacking RAD6 epistasis group genes, confirming that the PRR pathway is required for mutagenesis.

There appear to be NER-dependent and NER-independent components of error-prone repair. First, the levels of both cross-link repair and mutagenesis are higher in recombination-deficient, but NER-proficient, strains than in NER-deficient strains, suggesting that NER is required for a portion of errorprone repair. Second, the mutation patterns are different; recombination-deficient, but NER-proficient, yeast had the same distribution as repair-proficient cells, with more mutations on the non-transcribed strand. In contrast, there was no strand bias in NER-deficient strains.

Higher frequencies of psoralen-induced mutations on the non-transcribed strand have previously been reported in yeast and mammalian cells (50–54). DNA adducts are preferentially removed from active genes by transcription-coupled repair (55). NER excises adducts more quickly from the transcribed strand than the non-transcribed strand (56), leading to more mutations on the non-transcribed strand. The mutagenic repair of triple helix-directed psoralen cross-links in yeast requires PRR and shows a partial dependence on NER, in agreement with the current study (53, 54). A pathway for the error-prone repair of cross-links that depends on transcription-coupled NER and the trans-lesion bypass polymerase {eta} has been described in mammalian cells (57, 58).

Cross-link Repair Model—We suggest the following model for psoralen cross-link repair in yeast (Fig. 3). Psoralen interstrand cross-links are acted on by NER to yield repair intermediates that can enter recombinational or mutagenic pathways. In the major pathway the NER intermediates are processed to DSBs during DNA replication; these DSBs are repaired by HR in an error-free manner, generating crossovers and gene conversions. This pathway requires the genes of the NER and HR epistasis groups. The PRR genes RAD5 and RAD6 also have a role either in generating the recombinogenic intermediate or in entry of the intermediate into HR. Alternatively, cross-links may enter two mutagenic pathways, both requiring genes of the PRR epistasis group. In the NER-dependent pathway the NER-generated intermediates are repaired by error-prone PRR, generating mutations. PRR can also act directly on psoralen cross-links with lower efficiency in NER-independent error-prone repair.



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  		FIG. 3.
Model for interstrand cross-link repair. Psoralen interstrand cross-links are acted on by NER to form excision repair intermediates. Passage through DNA replication results in DSBs, which are repaired by HR. Mutagenic repair occurs by NER-dependent or NER-independent error-prone pathways, both of which depend on PRR.

 

   FOOTNOTES
 
* This work was supported in part by grants from the City University of New York PSC-CUNY research award program (to W. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Queens College, City University of New York, Dept. of Chemistry and Biochemistry, 65-30 Kissena Blvd., Flushing, NY 11367. Tel.: 718-997-4195; Fax: 718-997-5531; E-mail: Wilma_Saffran{at}qc.edu.

1 The abbreviations used are: NER, nucleotide excision repair; HR, homologous recombination; DSB, double strand break; PRR, post-replication repair. Back


   ACKNOWLEDGMENTS
 
We thank Errol Friedberg, Louise Prakash, Rodney Rothstein, and Lorraine Symington for providing yeast strains and plasmids. Olayemi Abioye, Judah Beck, Junyu Chen, Moshe Fink, Sabine Mayard, Hang Kyu Park, Sotirios Papantonis, and Rehan Taqi assisted with the genetic and physical analysis.



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