PCNA activates the MutLγ endonuclease to promote meiotic crossing over

Abstract

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..

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation

“…3B), consistent with the lack of visible co-localization of Mlh3 and Msh4 foci. This Mlh3-Msh4 interaction, even transient in vivo, supports recent in vitro data showing that the activity of the human MutLγ complex is strongly stimulated by both Exo1 and MutSγ (41,42).…”

Somatic CAG expansion in Huntington’s disease is dependent on the MLH3 endonuclease domain, which can be excluded viaMLH3splice redirection to suppress expansion

“…As MSH3, but not MSH6, is required for trinucleotide repeat expansion (16), MutSβ-stimulation of MutLγ endonuclease may be integral to repeat expansion in vivo . Other protein-protein interactions have been implicated in MutLγ endonuclease activation (63–65) and, interestingly, S. cerevisiae Mlh3 separation of function mutations provided evidence that distinct protein interactions may be required for MutLγ endonuclease activity in MMR and in meiosis (63, 64). Activity appears to be variably stimulated by proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), depending on the assay system (47, 50, 63, 65).…”

“…Other protein-protein interactions have been implicated in MutLγ endonuclease activation (63–65) and, interestingly, S. cerevisiae Mlh3 separation of function mutations provided evidence that distinct protein interactions may be required for MutLγ endonuclease activity in MMR and in meiosis (63, 64). Activity appears to be variably stimulated by proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), depending on the assay system (47, 50, 63, 65). Studies in S. cerevisiae have also indicated that Mlh3 endonuclease creates DNA breaks in the presence of RNA/DNA hybrids (R-loops) that can lead to increased instability, suggesting a link with transcription (66), though the relevance to repeat expansion in vivo is unclear.…”

Coordinated and Independent Roles for MLH Subunits in DNA Repair

“…In the major CO pathway, extended D-loops are stabilized by the meiosis-specific MSH family complex MSH4-MSH5 along with other members of the functionally diverse ZMM family, including ZIP1-4, MER3, and SPO16 (reviewed in [63]). This stabilization promotes the formation of double Holliday junctions; MLH1-MLH3 is thought to be recruited to ZMM-stabilized double Holliday junctions, where it asymmetrically cleaves them into only crossover products, potentially through interactions with PCNA [64][65][66][67] (Class I COs; Figure 3A). Importantly, the Class I CO pathway exhibits interference, which results in COs that are widely and evenly spaced, as well as CO assurance, the phenomena in which each homolog pair receives at least one CO.…”