Mutation Of MLH3 Endonuclease Motif Reveals Integration Between Crossover Pathways In Mammalian Meiosis

SUMMARY The MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that disrupts its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the null, but also results in male infertility. ABSTRACT During meiotic prophase I, double strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse Mlh3DN allele harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3−/− males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike Mlh3−/− males, however, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, DSB repair and CO designation factors persist in Mlh3DN/DN males, indicating a temporal delay in repair events. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3−/− males (10%), suggesting that the allele may be partially functional or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males, with evidence favoring the latter option. Double mutant mice that are homozygous for the Mlh3DN/DN mutation along with a null allele of Mus81, show losses in chiasmata approaching levels observed in Mlh3−/− males, suggesting that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in the Mlh3DN/DN spermatocytes. These results demonstrate that an intact MLH3 endonuclease domain is essential for most COs in mammalian meiosis, and that an endonuclease-impaired MutLγ retains the ability to facilitate the recruitment of other repair pathways, including MUS81 -EME1.