The suppression of COs during HR repair is imperative for the prevention of deleterious genomic rearrangements when non-sisters recombine. The DNA helicase Mph1 has been shown to negatively regulate the formation of COs during HR repair of a genomic DSB (4
). Here, we have confirmed this function of Mph1 in a plasmid break repair assay, indicating that Mph1 plays a core role in regulating CO formation during both chromosomal and extra chromosomal HR repair. Consistent with this notion is the ability of the S. pombe
Mph1 homolog, Fml1, to suppress CO formation in a plasmid gap repair assay (38
). Moreover, we have shown that the anti-CO function of Mph1 is absolutely dependent on MutSα. Previous studies have implicated MutSβ, but not MutSα, in the formation of COs during inter-chromosomal recombination (24
). However, our results indicate that during HR repair, a subset of COs are indeed generated in a MutSα-dependent manner but that the formation of these COs is antagonized by the actions of Mph1. This would explain why losing Msh6 alone has no effect on CO frequency since MutSα-dependent COs will normally be suppressed in wild-type cells by Mph1 (A). In contrast, Sgs1 did not suppress MutSα-dependent COs. Sgs1 has been shown to cooperate with MutSα in other forms of HR repair such as SSA, indicating HR pathway-specific interactions between Mph1, Sgs1 and MutSα (39
). MutSα-dependent COs that are suppressed by Mph1 do not require the presence of sequence divergence between the recombining sequences or, indeed, the mismatch recognition function of MutSα (C). This suggests that Mph1 is recruited to HR intermediates through a constitutive function of MutSα. Consistent with this idea is the finding that Mph1 and Msh6 are found to physically interact in undamaged cells (40
In contrast to the antagonistic interaction observed between Mph1 and MutSα when a completely homologous sequence was used to target repair, Mph1, together with Sgs1, was required for the efficient MutSα-dependent suppression of COs during homeologous recombination repair of pADE2 (1 bp/mis) (). This observation is consistent with recent findings that show Mph1 and Sgs1 can suppress chromosomal rearrangements mediated through non-allelic homeologous loci (41
). As expected, CO suppression during pADE2 (1 bp/mis) repair required the MMR functions of MutSα. However, our results did not reveal an overt defect in MMR in mph1Δ sgs1Δ
double mutant cells but rather indicated that in the absence of both Mph1 and Sgs1, NCOs are generated differently to when either or both helicases are present ( and ). NCOs generated by SDSA or double HJ dissolution tend to have short or undetectable GC tracts. This is because SDSA and dissolution require the disruption of D-loops and convergent double HJ branch migration, respectively, which limits the length of heteroduplex DNA and thus reduces the potential for mismatches distal to the break initiating MMR and causing GC (). Conversely, NCOs generated by HJ resolution would be expected to have the GC profile of CO products, as the two products result simply from HJ resolution occurring in alternative orientations (). The finding that the GC profile of NCOs was unaffected in mph1Δ
cells supports the notion that, in the absence of Mph1 or Sgs1, which results in compromised SDSA and dissolution, respectively, the majority of NCOs are generated by the remaining, intact NCO pathway. However, the altered GC profile of NCOs from mph1Δ sgs1Δ
double mutant cells compared to wild-type or single mutant mph1Δ
cells is consistent with a higher proportion of NCO products generated via HJ resolution. We also found that Mph1 had no effect on GC in CO products whereas the loss of Sgs1 gave rise to an increase in GC, as has previously been reported (34
). This observation is wholly consistent with a role for Mph1 in SDSA, because, in the absence of SDSA the channeling of intermediates into dissolution/resolution pathways of HR would be expected to affect the quantity of COs but not the GC profile of COs (). These results thus provide in vivo
evidence to support the proposed roles for Mph1 and Sgs1 in SDSA and dissolution, respectively and support the notion that, in mitotic HR repair, HJ resolution predominantly occurs as a back-up pathway when SDSA and dissolution are attenuated.
In conclusion, we have shown that the anti-CO functions of Mph1 are intricately linked to the MMR factor MutSα. A model outlining how we propose Mph1, Sgs1 and MutSα interact to regulate CO formation during homologous and homeologous recombination is shown in . The nature of these interactions is highly dependent on the nature of the recombining sequences since MutSα can promote (in the presence of homologous sequences) or suppress (in the presence of homeologous sequences) the formation of COs during the HR repair of DSBs. We propose that this latter role of MutSα, which requires its MMR function, acts to suppress the formation of double HJs during homeologous recombination by acting on the increased tracts of mismatches that are generated through Rad51-mediated D-loop extension and second end capture (, yellow box). Mph1 may thus promote MutSα-dependent suppression of double HJ formation by its ability to disrupt D-loops, thus circumventing the generation of mismatches. This proposal would explain why Mph1 is required for MutSα-dependent homeology-mediated suppression of COs without itself being a core component of the MMR machinery () (4
). In contrast, we propose that the MMR-independent functions of MutSα are required for the processing of double HJs into CO products and that this step is specifically inhibited by Mph1 but not Sgs1 (, green box). Our results therefore suggest that MutSα has multiple, separable functions in MMR and HR. Such a situation exists for MutSβ whereby mutant alleles of MSH2
indicate that the removal of non-homologous tails, and, heteroduplex rejection during SSA, are separable functions of MutSβ (42
). In addition to having a binding preference for mismatch-containing DNA, MutSα also binds HO-induced DSBs and synthetic HJs (43–45
). Such activities may be relevant to the pro-crossover function of MutSα during HR repair of DSBs and could be reminiscent of the function of the MSH4/MSH5 complex, which does not have a role in MMR but can bind HJs and has pro-crossover functions during meiosis (46–48
). How Msh6 performs functions aside from its role in mismatch recognition, and the molecular basis of how Mph1 and MutSα might cooperate to recognize and process recombination intermediates that do not contain base–base mismatches, are currently under investigation.
Mutations in the human homologues of Mph1, Msh2, Msh6 and Sgs1 (FANCM, hMSH2, hMSH6 and BLM, respectively) give rise to cancer-prone disorders that are associated with aberrant HR and genome instability (49–56
). Our findings, which were derived in haploid strains of S. cerevisiae
, are likely to be highly relevant to human cells where the potential for ectopic recombination and thus the necessity to suppress CO formation will be greater given the diploid and repetitive nature of the human genome.