Mechanism ofin vivoactivation of the MutLγ-Exo1 complex for meiotic crossover formation

Abstract

22Crossovers generated during the repair of programmed double-strand breaks 23 (DSBs) are essential for fertility to allow accurate homolog segregation during the 24 first meiotic division. Most crossovers arise through the asymmetric cleavage of 25 double-Holliday junction (dHJ) intermediates by the MutLγ endonuclease (Mlh1-26 Mlh3) and an elusive non-catalytic function of Exo1, and require the Cdc5/PLK1 27 kinase. Here we show in budding yeast that MutLγ forms a constitutive complex 28 with Exo1, and in meiotic cells transiently contacts the upstream MutSγ (Msh4-29 Msh5) heterodimer. Once recombination intermediates are committed to the 30 crossover repair pathway, MutLγ-Exo1 associates with sites of DSB hotspots, and 31 Exo1 recruits Cdc5 through a direct interaction that is required for activating 32 MutLγ and crossover formation. Exo1 therefore serves as a non-catalytic 33 Manuscript and Figures 2 matchmaker between Cdc5 and MutLγ. We further show that in vivo, MutLγ 1associates with the vast majority of DSB hotspots, but at a lower frequency near 2 centromeres, consistent with a strategy to reduce at-risk crossover events in these 3 regions. Our data highlight the tight temporal and spatial control of the activity of 4 this constitutive, potentially harmful, nuclease. 5 6 Introduction 7

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination

“…Indeed, homodimers of yeast Mlh1 have been reported, and an increase of their formation can inhibit MMR (74). In addition, in support of a possible heterodimer formation, a recent study in budding yeast found co-immunoprecipitation of Mlh3 with Pms1 (75). The crystal structure of the C-terminal region of human MLH1 (Protein Data Bank code 3RBN) showed that human MLH1 could form homodimers with the same residues involved in the heterodimer formation (14).…”

The Msh5 complex shows homeostatic localization in response to DNA double-strand breaks in yeast meiosis

“…Similarly in Sordaria, MSH4 foci appear in early meiotic prophase than MLH1 foci and the number of MSH4 is higher than that of MLH1 (Storlazzi et al, 2010). Interestingly, a recent study showed a chromosomal localization of a tagged version of Mlh1 in the budding yeast and the number of Mlh1 foci is less than ZMM foci in wild type, supporting a regulatory transition from MutSγ to MutLγ is operating in yeast meiosis (Sanchez et al, 2020).…”

Expanded roles for the MutL family of DNA mismatch repair proteins

“…It is not fully understood what kinds of chromosomal architecture or protein–protein interactions are required to stabilize Mlh1–Mlh3 or direct its function (Manhart et al, 2017). There is clear evidence for posttranslational modifications regulating meiotic crossover formation (He et al, 2020; Hollingsworth & Gaglione, 2019; Sanchez et al, 2020; Sourirajan & Lichten, 2008; Wild et al, 2019; Wild & Matos, 2016). For example, the structure‐specific endonucleases Mus81–Mms4 and Yen1 that act in the interference‐independent crossover pathway are activated and inhibited, respectively, by phosphorylation (Figure 1; Matos, Blanco, Maslen, Skehel, & West, 2011; Wild & Matos, 2016).…”