Distinct and overlapping roles of MutLγ, Mus81-Mms4, and STR in meiotic Holliday junction processing.

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.