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Molecular and Cellular Biology, March 2005, p. 2297-2309, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2297-2309.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DNA Interstrand Cross-Link Repair in the Saccharomyces cerevisiae Cell Cycle: Overlapping Roles for PSO2 (SNM1) with MutS Factors and EXO1 during S Phase

Louise J. Barber,¹ Thomas A. Ward,² John A. Hartley,¹ and Peter J. McHugh^2*

Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, University College London, London,¹ Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom²

Received 20 October 2004/ Returned for modification 16 November 2004/ Accepted 2 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pso2/Snm1 is a member of the ß-CASP metallo-ß-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G₂, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA interstrand cross-links (ICLs) are critical cytotoxic lesions (52), since they obstruct essential cellular processes such as transcription and replication. ICLs are produced by many commonly used anticancer agents, and there is growing evidence that acquired drug resistance may be due in part to enhanced repair of ICLs (19, 44).

ICL repair in eukaryotes is poorly understood, but several main steps have been identified, and others have been suggested to occur by analogy with the well-characterized ICL repair pathways in Escherichia coli (19). Repair is initiated through incisions made by the nucleotide excision repair (NER) apparatus (or, in the case of mammalian cells, possibly by a specialized reaction requiring XPF-ERCC1 [44]) that release the ICL, leaving an adducted oligonucleotide tethered to the opposing strand (sometimes referred to as the "uncoupling" or "unhooking" reaction) (12, 61, 70). Homologous recombination plays a role in ICL repair in both eukaryotes and prokaryotes (12, 17, 30, 43, 61). Biochemical reconstitution using purified E. coli proteins suggests that recombination into the resected post-NER gap provides the genetic template needed for a further round of incisions that remove the tethered oligonucleotide (61). DNA synthesis and ligation then restore the double helix. In contrast to that in bacteria, ICL processing is associated with double-strand break (DSB) formation in all eukaryotic systems examined to date (1, 15, 17, 43). Although the origin of these DSBs is not clear, they are associated with replication, and recent cellular and biochemical studies suggest that the collapse of a replication fork in the region of an ICL precipitates DSB formation (1, 4, 17, 43).

The Saccharomyces cerevisiae PSO2/SNM1 gene was identified in genetic screens for novel genes involved in the repair of ICLs produced by psoralen-UVA and nitrogen mustard (HN2), respectively (29, 56, 57). S. cerevisiae cells defective in PSO2 are uniquely sensitive to ICL-forming agents (including HN2, cisplatin, mitomycin C, and psoralens) but demonstrate wild-type resistance to monofunctional alkylating agents and ionizing radiation (57). Meiotic DSB processing appears unaffected in pso2/pso2 diploids, since spore viability appears wild type (29). Furthermore, pso2 mutants exhibit mild sensitivity to UVC, plausibly as a result of the minor ICL lesions produced (10). A mouse SNM1^–/– disruptant was sensitive to mitomycin C but not to a variety of other cross-linking drugs, suggesting that the function of this gene is partly conserved in higher eukaryotes (18). Despite the fact that PSO2/SNM1 was identified more than 20 years ago, very few clues as to its function have emerged.

Although PSO2 is epistatic with genes of the RAD3 (NER) epistasis group for ICL sensitivity (28), fundamental distinguishing features are apparent upon physical analysis. Following exposure to 8-methoxypsoralen-UVA, cisplatin, and nitrogen mustard, a complete abolition of cross-link incision events was observed in NER-defective strains (30, 39, 47, 71). In contrast, pso2 cells incised the cross-links normally, and both single- and double-strand break intermediates were formed (27, 39, 71). However, these were not subsequently reconstituted to intact double-stranded DNA (35, 39, 71). Hence, pso2 mutants are proficient at the initial "uncoupling" of ICLs but are unable to process the resulting DSB intermediates. Nevertheless, genetic analysis of the interaction of PSO2 with other repair genes failed to show epistasis with members of the RAD52 homologous recombination pathway (28, 35) required for DNA DSB repair following exposure of cells to ionizing radiation.

The Pso2 protein is a member of the ß-CASP metallo-ß-lactamase superfamily of enzymes, which share a hydrolytic domain similar to that of the mRNA cleavage and polyadenylation specificity factor, CPSF (8). One of the three human PSO2/SNM1 paralogues, Artemis/hSnm1C, was identified as the gene mutated in RS-SCID (radiation-sensitive severe combined immune deficiency), where the phenotype results from defects in V(D)J recombination and in the nonhomologous end joining (NHEJ) of DSBs (49). Recent in vitro analysis demonstrated that, alone, Artemis is a single-strand DNA-specific 5'-to-3' exonuclease, but when complexed with DNA-dependent protein kinase catalytic subunit, it becomes phosphorylated, acquiring 5' and 3' overhang and hairpin endonuclease activities (38).

The presence of a highly conserved hydrolytic metallo-ß-lactamase domain suggests a nucleolytic role for Pso2, by analogy with Artemis and CPSF. It is therefore possible that Pso2 acts on the DNA structures arising from NER-dependent ICL incision reactions in a nucleolytic fashion, providing a suitable substrate for recombination. To explore this hypothesis, we conducted a genetic study of the interactions of PSO2 with several key repair and recombination nucleases. We have discovered a functional overlap of the activity of Pso2 with those of the exonuclease 1 (Exo1) and MutS mismatch repair (MMR) factors during ICL repair in dividing cells, which appears to be restricted to S phase. We conclude that both NER and a Pso2-MutS-dependent pathway are required to process ICLs in S-phase cells and that this occurs prior to the repair of the DSBs induced in this phase of the cell cycle. Our results suggest that the primary role of homologous recombination, in response to ICLs, is to repair the DSBs generated at collapsed forks.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals and enzymes. Analytical-grade mechlorethamine (nitrogen mustard, or HN2) was purchased from Sigma Chemical Co. (Poole, United Kingdom).

Yeast strains. The yeast strains used in this study were derived from either B356-7C (a gift from L. Symington) or BY4741 (EUROSCARF Yeast Deletion Project). Genes were disrupted by PCR-based microhomology-targeted gene deletion using the pFA6 series of vectors and a plasmid-borne (pYES) copy of the URA3 gene to construct disruption cassettes (24, 37, 40). Disruptant strains are described in Table 1. Deletion was confirmed by PCR and further verified by restriction enzyme digests to yield a characteristic pattern of DNA fragments. Deletion primer sequences are available upon request.

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TABLE 1. Yeast strains used in this study

G₁ arrest using mating factor. Cells were incubated with -factor at 2 µg/ml in yeast extract-peptone-dextrose (YEPD) at 30°C for 2 h. A cross-linking drug was added to the arrested cultures, and cells were incubated for 30 min at 30°C on an orbital shaker. Then the cells were washed twice with phosphate-buffered saline (PBS) and diluted for plating on YEPD. Synchronization in G₁ was confirmed by fluorescence-activated cell sorting (FACS) for pilot experiments and by microscopy for all other experiments.

G₂ arrest using nocodazole. Cells were incubated with nocodazole at 15 µg/ml in YEPD for 1.5 h at 30°C; a fresh aliquot was then added, and cells were incubated for a further 1.5 h. Cross-linking drug was added to arrested cultures, and cells were incubated for 30 min at 30°C on an orbital shaker. Then the cells were washed twice with PBS and diluted for plating on YEPD. Synchronization in G₂ was confirmed by FACS for pilot experiments and by microscopy for all other experiments.

Experiments with release into S phase. Cells were incubated with -factor at 2 µg/ml in YEPD at 30°C for 2 h. Synchronization in G₁ was confirmed; then cells were washed twice with YEPD, resuspended in YEPD, and incubated at 30°C for 10 min prior to drug treatment. The drug was added to the released cultures, and cells were incubated for 30 min at 30°C on an orbital shaker. Then the cells were washed twice with PBS and diluted for plating on YPD. Analysis by FACS and scoring of bud emergence indicated that half the cells had entered S phase at the point of drug treatment.

Nitrogen mustard sensitivity assays. Nitrogen mustard sensitivity assays and determination of forward mutation rates were performed as previously described (42).

Determination of spontaneous mitotic recombination frequency and recombination classes. Single pink colonies were picked from strains grown on YEPD for 3 days at 28°C (growth for a longer period produces artificially high recombination rates, because Ade⁺ cells continue to divide longer than Ade^– cells) and were resuspended in 1 ml of water. Two hundred cells were plated in duplicate on synthetic complete-Trp medium to determine the total cell number. Appropriate dilutions (typically 2 x 10⁴ to 2 x 10⁷ cells/ml) were plated in duplicate on synthetic complete-Ade medium in order to determine the number of Ade⁺ prototrophs. Plates were incubated for 3 days at 28°C and then scored. Mean mitotic recombination rates were determined from the ratio of Ade⁺ prototrophs to the total cell number, as described previously (55). For each strain listed in Table 1, 20 individual Ade⁺ recombinants were analyzed for the recombination outcome. The integrated ade2-5'-TRP1-ade-n construct was amplified from these single colonies by PCR using primers flanking the rearranged region and was then digested with NdeI. The expected band sizes were 3.8 kb for the original construct, 2.1 and 1.7 kb for gene conversions, 2.77 kb for crossovers, and 1.7 and 1.06 kb for gene conversions associated with crossovers. PCR primer sequences are available upon request.

Pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis assays for DSB repair were performed as described previously (43).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synergistic increase in the cross-link sensitivity of pso2 cells in combination with exo1 and msh2. PSO2 is a member of the ß-CASP metallo-ß-lactamase family of enzymes, which typically possess hydrolytic nuclease activity (8). Consequently we examined the genetic interactions of PSO2 with other yeast nucleases that have a key role in DNA repair and/or recombination. We generated disruptants of pso2 in combination with rad27, the yeast homologue of mammalian FEN1 (25) involved in Okazaki fragment maturation and base excision repair; rad1, which produces the 5' incision during NER (16) and is also involved in the single-strand annealing recombination pathway (21); exo1, a 5'-to-3' nuclease with roles in homologous recombination and MMR (67, 68); and mre11, an endo/exonuclease which, in a complex with Rad50 and Xrs2, operates in homologous recombination (6) and the S-phase checkpoint (14). These strains were tested for sensitivity to the cross-linking drug nitrogen mustard (Fig. 1). We found that rad1 demonstrated an epistatic interaction with pso2 (Fig. 1A). The epistasis of rad1 and pso2 is like that reported for other NER mutants tested (rad4 and rad14 [data not shown] [30, 39]) and suggests that Rad1 does not play a role additional to its known function in NER-mediated ICL incision during ICL repair. The additive increase in sensitivity in the pso2 rad27 strain (Fig. 1B) suggests that Rad27 and Pso2 act on different substrates or intermediates during ICL repair, possibly consistent with a role for Rad27 in the base excision repair of monoalkylation damage produced by nitrogen mustards (42, 43) but perhaps also reflecting a role for Rad27 in some ICL-associated recombination events. For mre11 (Fig. 1C), we found a nonepistatic interaction with pso2 (similar to that observed between pso2 and rad52 group genes [Fig. 3] [26]) consistent with the previously observed sensitivity and DSB repair defect in mre11 strains (43). Most strikingly, pso2 shows a synergistic increase in sensitivity to this cross-linking drug in combination with exo1 (Fig. 1D). The exo1 single mutant shows no greater sensitivity than the parental strain, but deletion of pso2 in combination with exo1 leads to a dramatic increase in sensitivity to HN2. This suggests that Pso2 and Exo1 may compete for their substrate. Given the epistasis of PSO2 and NER genes for ICL repair, it was important to determine the relationship of NER genes to EXO1. Figure 1E demonstrates that deletion of exo1 in a rad4 background does not alter the sensitivity of the rad4 mutant. Identical results were obtained when rad1, exo1, and rad1 exo1 strains were compared (data not shown).

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FIG. 1. Analyses of the sensitivities of combinations of pso2, rad1, rad4, rad27, mre11, exo1, and msh2 gene disruptions (derived from B356-7C) treated with HN2. All results are means from at least three independent experiments. Error bars, standard errors of the means.

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FIG. 3. Relative sensitivities of disruptant strains (derived from B356-7C) to 100 µM HN2 added in G1, S, or G2 phase. All results are means from at least three independent experiments. Error bars, standard errors of the means.

EXO1 is known to interact genetically and physically with components of the MMR system, notably MSH2 and MLH1 (62, 68). A recent study has shown that Msh2, as part of the MutS complex, acts to regulate the 5'-3' exonucleolytic activity of Exo1 during MMR (23). Exo1 is known to be required for the processing of DSBs (69), and it has also been suggested that the Msh2-Msh3 (MutSß) heterodimer is recruited to DSBs and branched DNA structures, with a possible role in the promotion of cleavage by the Rad1-Rad10 endonuclease (20, 63). Consequently, we generated pso2 msh2 and exo1 msh2 double disruptants for analysis (Fig. 1F). The pso2 msh2 strain behaved indistinguishably from the pso2 exo1 strain, producing a synergistic increase in sensitivity to nitrogen mustard, whereas no increase in sensitivity was seen for an exo1 msh2 double disruptant. The level of sensitivity of the pso2 exo1 msh2 triple disruptant strain is very slightly greater than those of the pso2 exo1 and pso2 msh2 strains, suggesting that MMR proteins might play a minor role in ICL repair in addition to the overlapping pathway between pso2 and exo1/msh2.

DNA cross-link-associated DSB repair defect in pso2 exo1 and pso2 msh2 cells. It is known that pso2-null cells, and cells with disabling mutations in the Pso2 metallo-ß-lactamase domain, demonstrate defects in the repair of DSB intermediates generated at ICLs (35). In the repair-proficient strain B356 (Fig. 2) or an exo1 or msh2 single mutant (data not shown), DSB repair begins within 4 h and the DSBs are almost completely resolved within 24 h (estimated at around 50% repair by integrated optical density of full-size chromosomal bands). As expected, in nitrogen mustard-treated pso2 cells, these DSBs are much less efficiently resolved (estimated at around 15% restoration of chromosomal bands). Strikingly, the pso2 exo1 and pso2 msh2 double mutants are more severely affected, with no repair detectable, and DSBs continue to accumulate over 24 h (Fig. 2). This indicates that the increased sensitivity of the pso2 exo1 and pso2 msh2 cells reflects an amplified repair defect in the double mutants associated with reduced DSB resolution. Note that slightly higher levels of DSBs accumulate in the pso2 exo1 msh2 triple disruptant than in the pso2 exo1 and pso2 msh2 double-mutant strains, in agreement with the slightly increased level of HN2 sensitivity of the former strain seen in Fig. 1F. Since the NER genes show epistasis with PSO2 for sensitivity to cross-linking drugs, we also determined whether NER-defective cells are able to repair these DSBs. We discovered that repair of these DSBs was essentially completely disabled in the rad4 strain (Fig. 2) (we have observed the same result for a rad1 strain [data not shown]), indicating that ICL processing by both NER and Pso2, or Msh2-Exo1, must occur prior to resolution of the DSB by homologous recombination. This is consistent with the recent novel observations by Niedernhofer and colleagues (51), who demonstrated that ICL incision by the structure-specific nuclease XPF-ERCC1 precedes the healing of S-phase-associated DSBs produced after treatment with mitomycin C.

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FIG. 2. Repair of DSBs in HN2-treated pso2, pso2 exo1, pso2 msh2, pso2 exo1 msh2, and rad4 cells. Exponentially growing cells were either treated with 50 µM HN2 for 1 h at 28°C or mock treated (U) with water and were subsequently allowed to repair in minimal medium for 2, 4, or 24 h. The U sample was allowed to repair for 24 h. Samples were analyzed on contour-clamped homogeneous electric field gels.

No evidence for a major DSB repair defect in pso2 msh2/exo1 cells. Our data indicate that in the presence of functional NER, pso2 and msh2-exo1 might contribute to a repair pathway initiated by NER and that this event is required for repair of the DSBs associated with ICLs. Because these DSBs are repaired by homologous recombination, it was important to test whether the pso2/msh2-exo1 pathway affects homologous recombination more directly. We tested the sensitivity of combinations of pso2, exo1, and msh2 disruptants to a wide variety of genotoxic agents to see if disabling these redundant activities revealed more-general repair defects. We observed near-wild-type sensitivity to the alkylating agent methyl methanesulfonate; to UVC; to hydroxyurea, which imposes replicative stress by depleting nucleoside precursors; and to spindle poisons (data not shown). However, we did observe a mild increase in sensitivity to ionizing radiation (around 10-fold [data not shown]) and hydrogen peroxide for pso2 exo1 and pso2 msh2 double disruptants but not for their respective single mutants. This sensitivity was, nevertheless, far less than that observed in typical recombination mutants such as rad51 and rad52 mutants. Furthermore, pso2 cells and pso2 exo1 double mutants were fully viable following induction of a single HO-induced DSB, again in contrast to typical homologous recombination mutants (data not shown). Together these findings suggest that there is no major role for PSO2 or for an overlap in PSO2 MSH2/EXO1 activity during repair of frank DSBs by homologous recombination.

Essential role for PSO2 in ICL repair in G₁- and G₂-arrested cells but overlapping role for PSO2-MSH2 in growing cells. Homologous recombination (RAD52 epistasis group) mutants lose their ploidy-related resistance to DNA-damaging agents that produce DSBs (9, 29). For example, unlike wild-type cells, recombination-defective haploids are no more resistant to ionizing radiation in the G₂ and M phases of the cell cycle, where a sister chromatid is available as a substrate for the recombinational repair of DSBs, than in G₁. In contrast, their recombination-competent counterparts can employ this substrate, and they show increased resistance to ionizing radiation in G₂/M compared to G₁. Therefore, to determine whether Pso2 and Msh2-Exo1 play a role in ICL-induced homologous recombination, we analyzed the sensitivities of these strains in different cell cycle phases. It is clear that wild-type cells are able to repair ICLs efficiently in all three phases of the cell cycle tested (G₁, S, and G₂), since the sensitivity of the cells does not differ to a great degree across these phases (Fig. 3). S- and G₂-phase rad52 cells demonstrated modestly greater sensitivity to HN2 than the wild-type parent, suggesting that homologous recombination is not a major ICL repair pathway in this cell cycle phase, even though a substrate (sister chromatid) is available. G₁-phase rad52 cells demonstrated no increase in HN2 sensitivity over that of the wild-type, as previously reported (43), indicating that homologous recombination is not involved in this phase. Furthermore, rad52 is epistatic with rad4 and pso2-msh2, indicating that it is not a major secondary repair pathway in S phase. This is perhaps not unexpected, since for much of S phase no recombination substrate is available in haploid cells. Cells in which pso2 was disrupted showed equivalent, high sensitivity to HN2 in both G₁ and G₂ (Fig. 3A and C, respectively), indicating a critical role in both these phases of the cell cycle, but less sensitivity in S phase, similar to that seen in asynchronous experiments (Fig. 1). The pso2 msh2 double mutants demonstrated high sensitivity exclusively in S phase, equivalent to that demonstrated by pso2 single mutants in G₁ and G₂, indicating that the overlap in these two factors is restricted to only one cell cycle phase. Furthermore, pso2 msh2 cells are epistatic with rad52 in S phase (Fig. 3B), indicating that the homologous recombination triggered by ICLs is indeed dependent upon ICL processing by these factors. Taken together with the gels in Fig. 2, this is suggestive of an overlap in pso2 and msh2 at a step in ICL processing that occurs prior to repair of DSBs by recombination in S phase. The secondary nature of homologous recombination in ICL repair, rather than involvement in a pathway analogous to that in E. coli, is emphasized not only by the modestly increased sensitivity of rad52 cells in G₂ (Fig. 3C) but also by the lack of epistasis of rad4 and rad52 as well as pso2 and rad52 cells in G₂, where a substrate (sister chromatid) is fully available. In the presence of functional NER and Pso2, recombination does not play a major role in ICL repair; it does so only when these pathways are inactivated. In fact, the epistasis analysis presented in Fig. 1 using exponential-phase cells is representative of a very complex set of relationships between these various pathways in different phases of the cell cycle.

Overlapping roles for PSO2 and EXO1 in repair of spontaneous damage. From the results described above, it appears possible that PSO2-MSH2/EXO1 and NER genes are required to repair ICL damage that collapses replication forks, leading to DSBs, prior to recombinational restart of these forks but that recombination per se is possibly not generally involved in ICL repair. We wondered whether this role of NER, PSO2, and MSH2/EXO1 extended to spontaneous damage. Using a well-characterized, chromosomally integrated inverted-repeat substrate that allows mitotic recombination rates to be determined (54, 55), we assessed levels of spontaneous homologous recombination for the same panel of cells considered in Fig. 1. The substrate consisted of a 5'-truncated ade2 allele placed in an inverted orientation to a full-length ade2 gene harboring a frameshift mutation in an NdeI site (Fig. 4A). Recombination rates were scored by visual inspection of colonies to determine the number of Ade⁺ prototrophs in the plated population. Gene conversions and crossover events, as well as gene conversions associated with crossovers, were scored. This system has the additional advantage that, by determining the arrangement of the ADE2 construct following a recombination event, the nature of the event (gene conversion, crossover, or gene conversion event associated with a crossover) can be determined. The recombination rate of the pso2 single disruptant was similar to that of the wild-type strain (564 x 10^–6 and 585 x 10^–6 event/cell/generation, respectively) (Fig. 4B), whereas the exo1 and msh2 single mutants showed slight decreases (400 x 10^–6 and 386 x 10^–6 event/cell/generation, respectively). In contrast, the pso2 exo1 and pso2 msh2 double disruptants demonstrated reductions in the recombination rate (64 x 10^–6 and 55 x10^–6 event/cell/generation, respectively), somewhat less than that seen in a rad51 mutant (126 x10^–6 event/cell/generation) but not as severe as that of a rad52 mutant, known to be essentially completely recombination defective in this assay (0.03 x 10^–6 event/cell/generation) (55) (Fig. 4B). The pso2 exo1 msh2 triple disruptant behaved similarly to the pso2 exo1 and pso2 msh2 double mutants, and the exo1 msh2 strain behaved epistatically to its cognate single mutants. The rad51 and mre11 mutants demonstrated a significant reduction in spontaneous recombination (150 x 10^–6 event/cell/generation), as previously reported for the associated genes rad50 and xrs2 (54), but these levels were not modified by deletion of pso2. Of the other mutants considered, no statistically significant change in the recombination rate was observed for the rad4 disruptant or the rad27 disruptant (data not shown), and these rates were not further reduced by deletion of pso2, indicating that the apparent overlap of PSO2 in repair is specific for EXO1-MSH2. Together these data suggest that PSO2 and MSH2/EXO1 might play a role in the processing of spontaneous lesions that collapse replication forks. Interestingly, no diminution of spontaneous recombination was seen upon deletion of rad4, suggesting that the lesions acted on by Pso2-Msh2/Exo1 in the unperturbed cell cycle are not also NER substrates.

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FIG. 4. (A) Map of the inverted-repeat substrate. (B) Mean recombination rates between the ade2 inverted repeats, determined from at least nine independent colonies per strain. Error bars, standard errors of the means.

We also examined the outcomes of the recombination events by amplification of the ade2-5'-TRP1-ade-n construct by PCR, followed by diagnostic restriction digestion with NdeI (Table 2) (see Materials and Methods for experimental details). The ratios of gene conversions (75%) to crossovers (16.7%) and to conversions associated with crossovers (8.3%) were reminiscent of those previously reported with this construct for the parental, wild-type strain (55). The pso2 single mutant also produced an excess of gene conversions over crossovers. However, the exo1 single mutant produced a highly elevated level of crossovers relative to conversions (70 versus 20%), and the pso2 exo1 double disruptant behaved similarly (61.5 versus 19.2%). Thus, crossovers appear to be favored during spontaneous recombination at this construct in exo1 mutants, and pso2 behaves epistatically in this regard. Analysis of a smaller collection of recombinant colonies (10 per strain) from msh2 and pso2 msh2 strains indicated that they behave similarly to the exo1 and pso2 exo1 strains, giving 70 to 80% crossovers and 10 to 20% gene conversions (Table 2).

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TABLE 2. Recombination events at an ade2 inverted repeata

Overlap of both MutS factors MSH3 and MSH6 with PSO2. Msh2 acts in a heterodimer with either Msh3 or Msh6 during nuclear mitotic MMR. The MutS complex (Msh2-Msh6) primarily mediates MMR of small insertions-deletions and single-base mismatches, whereas MutSß (Msh2-Msh3) may preferentially act on large insertion-deletion loops (31, 33). Moreover, the Msh2-Msh3 heterodimer, along with the Rad1-Rad10 structure-specific nuclease, is involved in the removal of nonhomologous tails arising from homologous-recombination reactions (53, 59, 63). It is clear that a complex consisting of more components than just Msh2 and Exo1 mediates the Pso2 overlap, since overexpression of MSH2 or EXO1 alone, or both simultaneously, from high-copy-number vectors failed to suppress the HN2 sensitivity of pso2 strains (data not shown). We therefore wished to test which Msh2 partner subunit was involved in the pathway overlapping with Pso2. The pso2 msh3 and pso2 msh6 double disruptants demonstrated increases in sensitivity to HN2, but these were less than that of pso2 msh2 cells (Fig. 5A and B). Therefore, neither Msh2-Msh3 nor Msh2-Msh6 complexes were uniquely essential in the overlapping reaction. Consequently, we generated a pso2 msh3 msh6 triple disruptant. This was an exact phenocopy of the pso2 msh2 double disruptant, showing a synergistic increase in sensitivity to HN2 (Fig. 5C) indistinguishable from that observed for the pso2 msh2 double disruptant. We conclude that the Msh2-Msh3 and Msh2-Msh6 complexes along with Exo1 are redundantly involved in the pathway that overlaps with Pso2, and it is quite possible that additional factors are also required. The downstream factors acting during "classical" mitotic MMR are not absolutely required in this overlapping pathway, since a pso2 mlh1 double disruptant demonstrates a milder phenotype for sensitivity to HN2 than the pso2 msh2 strain (Fig. 5D).

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FIG. 5. HN2 sensitivities of pso2, msh3, msh6, and mlh1 strains and cognate multiple disruptants (derived from B356-7C) treated with HN2.

No evidence for a role for Pso2 in NHEJ. The human and murine Pso2-related factor Artemis is known to play a role in V(D)J recombination, a form of NHEJ (38, 49). We determined whether pso2 mutants show defects in this minor DSB pathway in yeast. This was achieved by transforming the strains with plasmid DNA (YEplac181) linearized in a region bearing no homology to the yeast genome with restriction enzymes producing a 5' (EcoRI) or 3' (PstI) overhang or incompatible ends (EcoRI and PstI double digestion). The ability to repair such DSBs following transformation into yeast cells, relative to that of a supercoiled control plasmid, has been widely used as a simple determinant of quantitative NHEJ efficiency and helped establish the roles of NHEJ factors such as Yku70/80 and Lig4 (5, 66). Neither the pso2, msh2, or exo1 single mutant nor the pso2 exo1 or pso2 msh2 double mutant exhibited a statistically significant reduction in the rejoining of these plasmids (data not shown). We used a ligase 4 (lig4) disruptant as a control for NHEJ deficiency in our experiments, and in agreement with the critical role of ligase 4 during NHEJ, the lig4 mutant exhibited a dramatically reduced ability to repair all types of DSBs. Therefore, we conclude that Pso2 is unlikely to play a major role, including a redundant role with Exo1 or Msh2, in NHEJ.

The overlap of PSO2 with EXO1 and MSH2 does not extend to MMR. Both MSH2 cells and EXO1 cells show defects in MMR. Consequently, we investigated whether the overlap in PSO2 activity with these factors extends to MMR. Using the canavanine resistance assay, we measured the forward mutation rates of a wild-type strain (BY4741) and its pso2, exo1, msh2, pso2 exo1, and pso2 msh2 derivatives. This assay is a highly sensitive marker for MMR defects (13). We found no significant differences in mutation rates between the wild type and the pso2 single disruptant (mean rates, 126 versus 209 mutants per 10⁸ survivors, respectively). The exo1 and msh2 mutants showed the expected elevated rates of forward mutation (1,750 and 2,754 mutants per 10⁸ survivors, respectively), but these rates were not further affected by deletion of pso2.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pso2 function overlaps with that of Exo1 and Msh2 in DNA repair. The work presented here is consistent with observations made over a 20-year period determining the genetic relationship of PSO2 with the other key ICL repair pathways identified to date: homologous recombination and NER (26, 30, 39, 43). The epistatic relationship between PSO2 and a variety of NER genes reported here and elsewhere (now including rad1, rad2, rad3, and rad4) suggests that these factors contribute to a common repair pathway (28). There is a clear ICL incision defect in NER mutants, not observed in pso2 mutants, placing Pso2 downstream of the incision step of NER (27, 39, 45, 71). As first reported in references 39 and 71, and substantiated here, pso2 mutants fail to repair the DSBs produced at ICLs. These DSBs are produced by replication fork collapse at ICLs, and their repair is controlled by homologous recombination (43). Consistent with a defect in the rejoining of these DSBs, decreases in mitotic recombination levels following ICL induction in pso2 mutants have been reported previously (58). Therefore, the processing of ICLs by Pso2 affects this DSB repair, but pso2 is, perhaps confusingly, not epistatic with rad52 in exponential-phase cells. Our results and those recently published by another group suggest an explanation for this. Niedernhofer et al. (51) demonstrated that the structure-specific nuclease XPF-ERCC1 is required for the repair of ICL-associated DSBs in mammalian cells. This nuclease is believed to act at the incision step of mammalian ICL repair, like the NER apparatus in yeast. The basis for the requirement for the whole NER apparatus in yeast versus XPF-ERCC1 in mammalian cells remains unknown. Here we obtained the strikingly similar result that yeast NER mutants are also unable to repair ICL-associated DSBs. Our data suggest that the processing of an ICL by NER and Pso2-MutS/Exo1 occurs prior to the repair of the collapsed-replication-fork-associated DSB and therefore is a prerequisite for the repair of ICL-associated DSBs. The epistasis of rad4 and pso2 msh2 with rad52 in S phase supports this hypothesis. The fact that these factors are not epistatic in G₂ cells suggests that, perhaps surprisingly, recombination is not the primary postincision/post-Pso2 pathway during ICL repair, even when a substrate is available.

We also found that combining a pso2 mutation with disruption of msh2 or exo1 leads to a significant (but certainly not complete) defect in spontaneous homologous recombination. Extrapolating from their role in the processing of ICLs prior to the restart of a broken replication fork, perhaps some form of endogenous damage resulting in an irreversibly collapsed replication fork must be processed by Pso2 and Exo1-MutS factors prior to recombinational replication restart. Many of the current models of fork restart are not applicable to forks encountering ICLs, because they are based on forks meeting an adduct that affects only one DNA strand (recently reviewed in reference 41). Therefore, a better understanding of the structures of repair forks inhibited by ICLs would help us understand the DNA structures acted upon by Pso2 and Msh2-Exo1 before the DSB and the fork are restored by recombination pathways. In this regard it is striking that mammalian cells often repair radiation-induced DSBs by NHEJ (36) but process ICL-associated DSBs almost exclusively by homologous recombination (7, 17). For example, CHO cells deficient in Ku80 (XRCC5 cells) show near-wild-type sensitivity to cross-linking drugs but are highly sensitive to ionizing radiation. In contrast, cells lacking XRCC2 and XRCC3 (Rad51 paralogues) are sensitive to ionizing radiation but also show very high sensitivity to cross-linking drugs and a profound defect in the repair of ICL-associated DSBs (17). This emphasizes the differing nature of the DSBs induced by total fork blockage versus genotoxic agents that directly break DNA. The lack of sensitivity of pso2 msh2/exo1 cells to an HO-induced DSB indicates that there is no defect in repairing DSBs per se. It is noteworthy, therefore, that apart from cross-linking drugs, the only other agents to which pso2 msh2/exo1 cells are sensitive are ionizing radiation and hydrogen peroxide. Notably, these both produce a huge range of chemically altered bases. It seems likely, therefore, that a subset of oxidized or modified bases produced by treatment with these agents are also substrates for Pso2 and MutS/Exo1. Interestingly, a role for the Arabidopsis thaliana homologue of PSO2 (AtSNM1) in the response to oxidative DNA damage has recently been reported (48).

Role for Pso2 and Exo1-Msh2 in influencing spontaneous recombination. The substrate employed in our recombination assays has been widely used to characterize recombination pathways, with the advantage that the nature of the recombination events affected can be readily identified through physical analysis of the products. Several models have been proposed to account for the spectrum of recombination events that can contribute to the recovery of ADE prototrophs following recombination events at this construct (64). Models of simple intrachromatid gene conversions, with or without associated crossovers, were initially used as explanations for these events (54, 55). Alternatives proposed more recently include misaligned sister chromatid exchanges, mediated by long conversion tracts, and break-induced recombination followed by single-strand annealing (64). The increased ratio of crossovers to gene conversions observed for the exo1 strain is consistent with a previous report of increased crossovers during gap repair in exo1 strains (65), although these were associated with an overall increase in recombination rates not observed in the present study. The authors of that report suggested that the increase in crossovers resulted from the elimination of MMR-mediated constraints on the recombination process, as seen in other studies (50, 60). Alternatively, our data could indicate that some intermediates generated during conversions are lethal in exo1 and msh2 strains and that cells generating these intermediates are consequently lost from analysis. In agreement with the results for the exo1 strain, we found that msh2 mutants behave similarly to exo1 strains, giving comparably elevated levels of crossovers. The codeletion of pso2 with either exo1 or msh2 does not seem to further alter the spectrum of events but does lead to an overall quantitative reduction. We suggest that this reduction is the result of decreased initial processing of some spontaneous lesions by Pso2 and Exo1-MutS complexes, and that this occurs prior to a subsequent recombination repair step perhaps restarting the fork blocked by this lesion. Therefore, it seems likely that Pso2 does not influence recombination itself and that Exo1 and MutS complexes have two roles in the processing of some spontaneous lesions that subsequently permit recombinational repair pathways, perhaps to restart broken replication forks. MutS complexes and Exo1 have an overlapping role with Pso2 in damage processing; then, once the processed intermediate enters a recombinational repair pathway, MutS and Exo1, as part of the MMR apparatus, can suppress crossovers during recombination. This might be achieved by rejection of pairing in the homeologous regions of DNA (20).

Roles of Pso2 and MutS-Exo1 in ICL repair. As outlined in the introduction, it is clear that in bacteria, yeast, and mammalian cells, an initiating step in ICL repair involves incisions, bracketing the ICL, that release the adduct on one strand (12, 17, 30). For E. coli and S. cerevisiae, this step appears to involve an essentially full complement of essential NER factors, Uvr A₂BC in E. coli (70) and at least Rad1, Rad10, Rad2, Rad3, Rad4, and Rad14 in the yeast, since all the corresponding yeast NER mutants demonstrate high sensitivity to cross-linking agents (30, 42, 47). Because it has been clearly established that, in contrast to NER-deficient cells, pso2 mutants incise ICLs normally, we can rule out a key role for Pso2 in the initial incision event. In Fig. 6 we outline a possible model to reconcile the observations made here, and elsewhere, concerning the roles of NER, Pso2/MutS-Exo1, and homologous recombination in ICL repair through the yeast cell cycle. In G₁ cells, NER genes and PSO2 are epistatic with each other, and deletion of msh2 does not further affect the viability of pso2 cells. This suggests that a pathway consisting of NER incisions followed by a Pso2-controlled step operates in this cell cycle phase. A homologous recombination mutant (rad52) displayed no increased sensitivity in G₁-phase cells, consistent with our previous report (43) using stationary-phase cells, suggesting that the homologous recombination apparatus plays no role in G₁ cross-link repair. Given the known sensitivity of rev3 mutants to ICL-inducing agents in stationary-phase yeast cells (43), it is possible that DNA polymerase is required to complete repair. Furthermore, rad52 does not appear to contribute to any salvage pathways in G₁ cells, since deletion of rad52 in NER-defective or pso2 cells does not lead to a significant increase in sensitivity. To our surprise G₂ rad52 cells were not highly HN2 sensitive compared to pso2 and NER mutant strains. Therefore, although it is clear that NER and Pso2 contribute to the major G₂ ICL repair pathway, rad52 apparently is not the main downstream repair mechanism employed. rad52 cells are more sensitive in G₂ than in G₁ and, in contrast to G₁ cells, an increase in the sensitivity of both NER-defective and pso2 cells is seen upon deletion of rad52 in G₂ cells. Therefore, a recombination-dependent ICL repair reaction does function when NER and Pso2 fail. We therefore propose that the major G₂ ICL repair pathway does not depend on recombination. We suggest that a minor recombination-dependent pathway operates which must also involve some type of initial ICL incision or nucleolytic processing to provide access to the ICL, and this can be NER independent.

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FIG. 6. Roles of NER, Pso2, MutS-Exo1 factors, and homologous recombination in processing of intermediates generated at ICLs in different phases of the yeast cell cycle. TLS, translesion synthesis; H.R., homologous recombination.

The situation in S-phase cells appears complex. The ICL will block replication forks and precipitate DSB formation. Both biochemical and cell biology evidence suggest that the formation of DSBs at ICLs during replication is NER independent (1, 4, 17, 43). ICLs will be incised by NER in order to initiate repair, since it is known that NER is required to incise ICLs throughout the cell cycle (46). The reduced sensitivity of pso2 mutants in S phase appears to result from the existence of additional competing processes associated with MMR factors and Exo1. It is, we would speculate, possible that association of MMR factors with PCNA (11, 22), and therefore the replicative apparatus (32), targets them to ICL intermediates during S phase and that this occurs in competition with Pso2. Note that this does not imply that the substrates acted on by Pso2 and MutS-Exo1 are the same. It is, for example, possible that MMR factors act on ICLs that are encountered by the replication fork, while Pso2 acts on the remainder. Furthermore, pso2 msh2 cells are more sensitive than NER mutant cells in S phase. It is possible that MMR factors and NER factors are both able to incise ICLs in S phase and that postincision processing occurs by Pso2 or Exo1, depending on the complex (NER or MMR) that makes the initial incision. However, the lack of an increase in sensitivity in rad4 and rad1 strains upon deletion of exo1 argues against the latter possibility. Furthermore, Beljanski and colleagues (3) recently studied the genetic interaction between rad1 and several MMR factors (msh6, pms2, and msh2) and demonstrated that they were clearly epistatic for HN2 sensitivity. Therefore, some yet-to-be-identified ICL processing pathway likely operates in S phase (perhaps associated directly with replication fork demise), generating a substrate for Pso2 and Msh2-Exo1. Biochemical analysis will be required to address these possibilities in detail, but unpublished data (from the Robb Moses laboratory) indicate that purified Pso2 is a potent 5'-3' double-stranded and single-stranded DNA exonuclease, clearly suggesting that the alternative targeting of Pso2 or Exo1 to ICL repair intermediates might well account for overlap in these factors. The recent reports that PCNA might be required to target MutS factors to psoralen ICLs in mammalian cell extracts, and that this influences an early stage in ICL repair (34, 72), also support this notion. In addition, our results and those of Niedernhofer and colleagues quite conclusively indicate that ICLs must be processed by NER incision and the action of Pso2 and/or MutS-Exo1 prior to DSB healing. Whether the ICL processing and DSB repair events are mechanistically coordinated is not known, but it is conceivable that the DSBs resulting from replication fork collapse are effectively secondary, less toxic lesions which require that the ICL must be fully repaired prior to its recombinational restart.

One difficult question arising from all the models presented here and elsewhere is the paucity of knowledge concerning the steps occurring after ICL incision. While it is formally possible that NER and Pso2 could alone achieve complete ICL repair, for example, in G₂ cells, this seems rather unlikely. Many previous models favor a recombination-dependent gap-filling step following ICL incision, analogous to the pathway established in E. coli, and it easy to imagine how this could operate in G₂ cells. However, our work does not support such a step in any phase of the yeast cell cycle. Although we do not at present understand the downstream steps of ICL repair, it seems likely that lesion tolerance by translesion polymerases and bypass DNA synthesis might be involved. Certainly polymerase -deficient cells are more sensitive to nitrogen mustard in stationary than in exponential phase (43), although it is not known whether the role of polymerase occurs post-ICL incision. We have gathered preliminary evidence that the components of the postreplication repair apparatus might make a major contribution to these postincision pathways, since rad6 and rad18 cells are highly sensitive to ICL-inducing agents throughout their cell cycle (T. Ward and P. J. McHugh, unpublished data). In fact, on this basis, we propose that a Rad6-Rad18-dependent tolerance mechanism acts after ICL incision in G₂ cells, and this mechanism could involve either translesion synthesis polymerases or a copy choice tolerance pathway (as shown in Fig. 6). Future studies will determine whether and how the various subpathways and polymerases controlled by RAD6-RAD18 contribute to the postincision steps of ICL repair.

 
We are very grateful to Lorraine Symington for the kind gift of several yeast strains. We also thank Ian Hickson, Sovan Sarkar, and Minoru Takata for comments on the manuscript. Robb Moses very kindly shared unpublished observations on the biochemical activity of Pso2.

This work was supported by Cancer Research UK and The Royal Society.

 
* Corresponding author. Mailing address: Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222-441. Fax: 44-1865-222-431. E-mail: peter.mchugh{at}cancer.org.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, March 2005, p. 2297-2309, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2297-2309.2005
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