A mutation in the endonuclease domain of mouse MLH3 reveals novel roles for MutLγ during crossover formation in meiotic prophase I

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 (MutL gamma). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3(DN/DN)) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3(DN/DN) males, like fully null Mlh3(-/-) males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3(-/-) males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutL gamma in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3(DN/DN) males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3(DN/DN) spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3(-/-) males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutL gamma-defined repair intermediates in Mlh3(DN/DN) males. Double mutant mice homozygous for the Mlh3(DN/DN) and Mus81(-/-) mutations show losses in chiasmata close to those observed in Mlh3(-/-) males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3(DN/DN) spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3(DN/DN) complex display the proper loading of factors essential for CO resolution (MutS gamma, CDK2, HEI10, MutL gamma). Despite these functions, mice bearing the Mlh3(DN/DN) allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. Author summary Meiosis is a specialized cell division whereby a diploid cell undergoes one round of DNA replication followed by two rounds of division, yielding up to four haploid gametes. This process depends on tethering of maternal and paternal homologous chromosomes, and by the formation of crossovers (COs) between homologs during prophase I. COs arise from programmed double-strand breaks (DSBs), and can form via one of at least two mechanisms (class I, class II). In mouse, class I represents the major CO pathway, with the MLH1-MLH3 (MutL gamma) complex being critical. MLH3 contains a conserved metal binding motif, DQHA(X)(2)E(X)(4)E, required for its endonuclease function, and this activity is postulated to represent a resolvaseactivity for class I COs. We generated a point mutant (Mlh3(DN)) in the endonuclease domain without altering the overall structure of MutL gamma. Mlh3(DN/DN) males have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and chromosome pairing. The MLH3(DN) mutation permits normal loading of MutL gamma, but key DSB repair factors persist in Mlh3(DN/DN) males, indicating a temporal delay in repair and suggesting a mechanism by which alternative DSB repair pathways may be selected. Thus, the endonuclease domain of MLH3 is important for normal processing of DSB repair intermediates.