Bloom's helicase (BLM) is thought to prevent crossing-over during DNA double-strand-break repair (DSBR) by disassembling double-Holliday junctions (dHJs) or by preventing their formation. We show that the Saccharomyces cerevisiae BLM ortholog, Sgs1, prevents aberrant crossing-over during meiosis by suppressing formation of joint molecules (JMs) comprising three and four interconnected duplexes. Sgs1 and procrossover factors, Msh5 and Mlh3, are antagonistic since Sgs1 prevents dHJ formation in msh5 cells and sgs1 mutation alleviates crossover defects of both msh5 and mlh3 mutants. We propose that differential activity of Sgs1 and procrossover factors at the two DSB ends effects productive formation of dHJs and crossovers and prevents multichromatid JMs and counterproductive crossing-over. Strand invasion of different templates by both DSB ends may be a common feature of DSBR that increases repair efficiency but also the likelihood of associated crossing-over. Thus, by disrupting aberrant JMs, BLM-related helicases maximize repair efficiency while minimizing the risk of deleterious crossing-over.

Summary Saccharomyces cerevisiae RecQ helicase, Sgs1, and XPF-family endonuclease, Mus81-Mms4, are implicated in processing joint molecule (JM) recombination intermediates. We show that cells lacking either enzyme frequently experience chromosome segregation problems during meiosis and when both enzymes are absent attempted segregation fails catastrophically. In all cases, segregation appears to be impeded by unresolved JMs. Analysis of the DNA events of recombination indicates that Sgs1 limits aberrant JM structures that result from secondary strand-invasion events and often require Mus81-Mms4 for their normal resolution. Aberrant JMs contain high levels of single Holliday junctions and include intersister JMs, multi-chromatid JMs comprising three and four chromatids, and newly identified recombinant JMs containing two chromatids, one of which has undergone crossing-over. Despite persistent JMs in sgs1 mms4 double mutants, crossover and noncrossover products still form at high levels. We conclude that Sgs1 and Mus81-Mms4 collaborate to eliminate aberrant JMs whereas as-yet-unidentified enzymes process normal JMs.

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.

During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the heterotetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51–single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues.