During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defects in crossing over cause infertility, miscarriage and congenital disease. Accordingly, each pair of chromosomes attains at least one crossover through processes that designate and then implement crossing over with high efficiency 2 . At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates [3][4][5] . A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 6 . Although the endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution [7][8][9][10][11][12] , the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp, PCNA, is important for crossover-biased resolution. In vitro assays with human enzymes show that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease under physiological conditions.In this context, the hMutLγ endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutSγ, the latter of which binds Holliday junctions 13 . hMutLγ also specifically binds a variety of branched DNAs, including Holliday junctions, but canonical resolvase activity is not observed implying that the endonuclease incises adjacent to junction branch points to effect resolution. In vivo, we show that budding yeast RFC facilitates MutLγdependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair [14][15][16] and evoke a novel model for crossover-specific dHJ resolution during meiosis..

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defects in crossing over cause infertility, miscarriage and congenital disease. Accordingly, each pair of chromosomes attains at least one crossover through processes that designate and then implement crossing over with high efficiency 2 . At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates [3][4][5] . A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 6 . Although the endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution 7-12 , the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp, PCNA, is important for crossover-biased resolution. In vitro assays with human enzymes show that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease under physiological conditions. In this context, the hMutLγ endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutSγ, the latter of which binds Holliday junctions 13 . hMutLγ also specifically binds a variety of branched DNAs, including Holliday junctions, but canonical resolvase activity is not observed implying that the endonuclease incises adjacent to junction branch points to effect resolution. In vivo, we show that budding yeast RFC facilitates MutLγdependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair [14][15][16] and evoke a novel model for crossover-specific dHJ resolution during meiosis. MainMeiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via homologous pairing and DNA strand-exchange to form joint-molecule (JM) intermediates 1 . Regulatory processes designate a subset of events to become crossovers, and at these sites nascent JMs are matured into double-Holliday junctions (dHJs). Through poorly defined mechanisms, MutLγ (comprising MLH1 and MLH3) accumulates at prospective crossover sites and biases dHJ resolution to specifically produce crossovers 17,18 . When MutLγ is dysfunctional, dHJs are still resolved, but with a non-crossover outcome 8 . Consequently, chromosome segregation fails and fertility is diminished. MLH1 and MLH3 are conserved members of the MutL family of DNA mismatch-repair (MMR) factors that couple mismatchrecognition by a MutS complex to downstream excision and resynthesis of the nascent strand 14,16 . MutL complexes from diverse species possess endonuclease activity that provides an initiation site for mismatch excision by a 5'-3 exonuclease such as EXO1 15 . During DNA replication, MutL-catalyzed incision, and thus MMR, is specifically targeted to the nascent strand via an orientation-specific...

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization.