Mechanism of in vivo activation of the MutL-Exo1 complex for meiotic crossover formation

Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination and their subsequent repair culminates in crossover (CO) formation. COs result from the asymmetric cleavage of double-Holliday junction (dHJ) intermediates, that requires the MutL? endonuclease and a non-catalytic function of Exo1, an activity essential for fertility but at risk of generating unwanted chromosome rearrangements. Here we show how crossover formation by MutL? is activated at the right time and at the right place. MutL? forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the upstream MutS? (Msh4-Msh5) heterodimer. MutL? associates with DSB hotspots only once recombination intermediates have been stabilized and engaged in the crossover repair pathway. MutL?-Exo1 is recruited to DSB hotspots independently of the polo-like Cdc5 kinase, but to activate dHJ resolution, Cdc5 is recruited to the recombination intermediates and interacts individually with both MutL? and Exo1, suggesting their direct modification. in vivo, MutL? occupancy is restrained on recombination intermediates, and MutL? associates with the vast majority of DSB hotspots, but at a lower frequency in centromeres, consistent with a strategy to reduce at-risk crossover events in these regions, and in late replicating regions. Our data highlight the tight temporal and spatial control of the activity of this constitutive, potentially harmful, nuclease. Overall design: Two biological replicate Spo11-oligo maps of Saccharomyces cerevisiae strain mlh3 at meiotic 5 hr time point