PCNA activates the MutLγ endonuclease to promote meiotic crossing over

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation¹. Defective crossing over causes 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. At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction (dHJ) intermediates^2,3. A central tenet of crossover resolution is that the two Holliday junctions (HJs) are resolved in opposite planes by targeting nuclease incisions to specific DNA strands⁴. The endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution^5–10, but 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 reveal that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease. In this context, hMutLγ is further stimulated by a co-dependent activity of pro-crossover factors hEXO1 and hMutSγ, the latter of which binds HJs¹¹. hMutLγ also specifically binds a variety of branched DNAs, including HJs, 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 MutLγ-dependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and the initiation steps of DNA mismatch repair^12,13 and evoke a novel model for crossover-specific dHJ resolution during meiosis.