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

Toledo, M.; Sun, X.; Brieno-Enriquez, M. A.; Raghavan, V.; Gray, S.; Pea, J.; Venkatesh, A.; Patel, L.; Borst, P. L.; Alani, E.; Cohen, P. E.

doi:10.1101/517748

bioRxiv · 10.1101/517748

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

Toledo, M.·Sun, X.·Brieno-Enriquez, M. A.·Raghavan, V.·Gray, S.·Pea, J.·Venkatesh, A.·Patel, L.·Borst, P. L.·Alani, E.·Cohen, P. E.

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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 (MutL{gamma}). 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 normal DSBs and undergo normal 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 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 MutL{gamma}-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata that approach levels observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed 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 (MutS{gamma}, CDK2, HEI10, MutL{gamma}). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. SUMMARYThe MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that alters its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the full null Mlh3 mouse, but also results in male infertility.

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Toledo, M., Sun, X., Brieno-Enriquez, M. A., Raghavan, V., Gray, S., Pea, J., Venkatesh, A., Patel, L., Borst, P. L., Alani, E., Cohen, P. E.. 2019-01-11. Mutation Of MLH3 Endonuclease Motif Reveals Integration Between Crossover Pathways In Mammalian Meiosis. https://doi.org/10.1101/517748

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