RPA directly stimulates Mer3 helicase processivity to ensure normal crossover formation in meiosis.

Meiotic crossover formation is critical for generating viable gametes and enhancing genetic diversity. The helicase Mer3 (HFM1 in humans) is a highly conserved factor essential for promoting crossovers and ensuring their proper distribution. Here, we identify replication protein A (RPA) as a direct interactor of budding yeast Mer3. We demonstrate that this interaction is conserved between human HFM1 and RPA. Cross-linking mass spectrometry and structural modelling with AlphaFold2 reveal a conserved and specific Mer3-RPA interface. Single-molecule magnetic tweezers assays demonstrate that direct RPA interaction is required for Mer3 helicase processivity under conditions of low DNA tension. Consistently, a mer3 mutant deficient in RPA binding exhibits reduced crossover frequencies and accumulates unresolved recombination intermediates during budding yeast meiosis. Via genome-wide localisation experiments, we link this effect to weakened recruitment of the mer3 mutant to double-strand break sites. Our findings provide mechanistic insights into coordination of meiotic recombination by the Mer3 helicase through interactions with the canonical DNA repair machinery, highlighting a conserved mechanism underlying crossover control during sexual reproduction.