Distinct and overlapping roles of MutLγ, Mus81-Mms4, and STR in meiotic Holliday junction processing
2026-06-02
Most meiotic crossovers arise from the nucleolytic resolution of recombination intermediates that ZMM proteins stabilize as double Holliday junctions (dHJs). MutLγ is the nuclease thought to resolve these ZMM-bound dHJs into crossovers, but alternative enzymes - including Mus81-Mms4 and the Sgs1-Top3-Rmi1 (STR) complex - can also process meiotic DNA joint molecules. How ZMM-bound dHJs are preferentially steered toward MutLγ-mediated processing has remained unresolved, in part because experimental systems have been unable to uncouple dHJ resolution from upstream recombination events and downstream cell-cycle progression. To overcome this limitation, we engineered a budding yeast system that stabilizes pre-existing ZMM-bound dHJs, eliminates the continued occurrence of upstream recombination events, and enables conditional pathway-specific resolution without cell-cycle advance. Using this approach, we show that MutLγ is uniquely capable of imposing crossover-specific resolution on ZMM-bound dHJs. In contrast, Mus81-Mms4 and STR can access crossover-designated recombination intermediates but generate mixed or exclusively noncrossover products. We further identify an Sgs1-independent role for Top3-Rmi1 in maintaining ZMM-dHJ architecture and preventing their conversion into aberrant, MutLγ-refractory species. Together, our findings reveal that ZMM proteins establish a hierarchy, rather than absolute selectivity, in dHJ processing, one that favours MutLγ-directed crossovers while preserving alternative resolution routes to safeguard chromosome segregation.