Evolutionary specialization of Rad51 and Dmc1 tune recombination outcomes

Homologous recombination (HR) relies on the RecA-family recombinases Rad51 and Dmc1, which both catalyze DNA strand pairing and exchange but promote distinct recombination outcomes. How these recombinases became functionally specialized remains unclear. Here, we combine phylogenetic analysis with functional assays to define the basis of this specialization. We identify conserved amino acid variation in the DNA-binding Loop 2 region across the archaeal– eukaryotic lineage and show that these residues tune recombinase interactions with double- stranded DNA. Guided by these evolutionary signatures, we engineer Rad51 variants with altered DNA-binding properties and test their functions. Rad51-like variants promote break-induced replication (BIR), whereas substitutions characteristic of Dmc1 suppress BIR and instead favor crossover outcomes, as measured using mitotic crossover assays. These results indicate that evolutionary divergence in Loop 2 alters recombinase binding on reaction intermediates, influencing their stability and progression toward distinct repair outcomes. Thus, evolutionary tuning of recombinase–DNA interactions shift the balance between recombination pathways, providing a mechanistic basis for the functional specialization of Rad51 and Dmc1 during mitotic and meiotic HR.