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 ( 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 MutLγ 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 MutLγ-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 (MutSγ, CDK2, HEI10, MutLγ). 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.

Summary Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, Sororin, and PDS5B and direct cleavage by Separase. Herein, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by the protein phosphatase PP1γ, which both interacts with, and is a phosphotarget, of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ leading to dephosphorylation of WAPL, which in turn results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.