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/DN) 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 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 Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. 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 permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/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 Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.
Highlights
The MLH3DN mutation permits normal loading of MutLγ, but key double-strand breaks (DSBs) repair factors persist in Mlh3DN/DN males, indicating a temporal delay in repair and suggesting a mechanism by which alternative DSB repair pathways may be selected
The endonuclease domain of MLH3 is important for normal processing of DSB repair intermediates
Meiosis is a specialized cell division process in which a diploid parental cell undergoes one round of DNA replication followed by two rounds of division, resulting in up to four haploid gametes
Summary
Meiosis is a specialized cell division process in which a diploid parental cell undergoes one round of DNA replication followed by two rounds of division, resulting in up to four haploid gametes. Successful halving of the genome during meiosis I depends on the tethering of maternal and paternal homologous chromosomes during meiotic prophase I, and their subsequent release at the first meiotic division. This tethering is ensured by homologous recombination, leading to the formation of crossovers; by synapsis, the formation of a tripartite proteinaceous structure, the synaptonemal complex, or SC between homologous chromosomes; and by cohesion between replicated sister chromatids that ensures appropriate tension on the metaphase I spindle [1,2]. In M. musculus, only 10% of DSBs are repaired as COs, while the majority are mostly repaired as NCOs, presumably via SDSA or other pathways, [6,7,8,9]
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