Mechanism of in vivo activation of the MutLgamma-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? complex together with 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 at a late step in the recombinational repair, 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 genome-wide, 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 highly temporally and spatially control of the activity of this constitutive, potentially harmful, nuclease Overall design: 9 samples total: 2 independent replicates of Mer3-Flag ChIP at 4 h in meiosis and their control untagged anti-Flag ChIP at 4 h in meiosis; 2 Mlh3-Myc8 ChIP at 5 h and 5h30 in pCUP1-IME1-synchronized meiosis and their corresponding controls spo11? Mlh3-Myc8 ChIP at 5 h and 5h30 in pCUP1-IME1-synchronized meiosis; 1 exo1? Mlh3-Myc8 ChIP at 5h30 in pCUP1-IME1 synchronized meiosis and its corresponding control spo11? Mlh3-Myc8 ChIP at 5h30 in pCUP1-IME1 synchronized meiosis.