PCNA activates the MutLγ endonuclease to promote meiotic crossing over

Abstract

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. 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 efficiency2. At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates3-5. A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands6. Although the endonuclease activity of the MutL{gamma} complex has been implicated in crossover-biased resolution7-12, 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 hMutL{gamma} endonuclease under physiological conditions. In this context, the hMutL{gamma} endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutS{gamma}, the latter of which binds Holliday junctions13. hMutL{gamma} 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 MutL{gamma}-dependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair14-16 and evoke a novel model for crossover-specific dHJ resolution during meiosis.