LAUSR

Significance Sexual eukaryotes fall into two groups with respect to their RecA-like recombinases. The first group possesses Rad51 (ubiquitous) and Dmc1 (meiosis-specific), which cooperate to conduct interhomolog recombination in zygotes with high sequence heterogeneity. Interestingly, Dmc1 was lost from the second group of eukaryotic organisms. Here we used the industrial workhorse fungus Trichoderma reesei to address if and how Rad51-only eukaryotes carry out hybrid meiosis. We show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and that TrRad51, like Saccharomyces cerevisiae Dmc1, possesses a better mismatch tolerability than S. cerevisiae Rad51. Our results indicate that the ancestral TrRad51 evolved to acquire Dmc1-like properties by adopting multiple structural variations in the L1 and L2 DNA-binding loops. Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.