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 MutLγ 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 MutLγ is activated at the right time and at the right place. MutLγ forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the upstream MutSγ (Msh4-Msh5) heterodimer. MutLγ associates with DSB hotspots only once recombination intermediates have been stabilized and engaged in the crossover repair pathway. MutLγ-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 MutLγ and Exo1, suggesting their direct modification. in vivo, MutLγ occupancy is restrained on recombination intermediates, and MutLγ 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.程序性DNA双链断裂（Programmed DNA double-strand breaks, DSBs）可启动减数分裂重组，其后续修复过程最终催生交换（crossover, CO）产物。交换由双霍利迪联结体（double-Holliday junction, dHJ）中间体的不对称切割产生，该过程依赖MutLγ核酸内切酶与Exo1的非催化功能——这一活性对生育力至关重要，但也存在引发意外染色体重排的风险。本研究阐明了MutLγ介导的交换形成如何在正确的时间与位置被激活：MutLγ与Exo1构成组成型复合物，在减数分裂细胞中会短暂结合上游的MutSγ（Msh4-Msh5）异二聚体；仅当重组中间体已稳定并参与交换修复通路时，MutLγ才会结合至DSB热点。MutLγ-Exo1可独立于polo样Cdc5激酶被招募至DSB热点，但要激活dHJ解离，Cdc5需被招募至重组中间体，并分别与MutLγ和Exo1相互作用，提示二者发生了直接修饰。体内实验显示，MutLγ在重组中间体上的结合占有率受到严格限制；尽管MutLγ可结合绝大多数DSB热点，但在着丝粒区域与晚复制区域的结合频率更低，这与该类区域通过减少潜在风险交换事件来规避风险的策略相符。本研究数据凸显了这一组成型、潜在有害的核酸酶的活性在时间与空间上的严格调控。