Rad54 separation of function mutation highlights unique roles during homologous recombination

Homologous recombination (HR) is a DNA repair pathway that utilizes a template-based approach to repair double-strand breaks within the genome. Template utilization requires the exchange of individual DNA strands, which members of the RecA family of recombinases facilitate. Rad51 is a primary strand exchange factor in eukaryotes. During regular mitotic DNA repair, Rad51 is aided by the DNA translocase Rad54, which acts as a motor to remodel the template DNA and stabilize primary-strand exchange intermediates. The regulation of this activity remains incompletely understood. Here, we have identified a conserved site within the C-terminal region of Rad54. The mutation of this site creates a functional separation at early strand-exchange intermediates in vivo. Using this mutant protein, we identify a novel intermediate essential for stabilizing displacement loop (D-loop) structures. This precedes the removal of Rad51 and DNA extension. Based on our experiments, we hypothesize that this Rad54 mutant cannot stabilize Rad51-mediated strand-exchange intermediates because it cannot topologically isolate dsDNA regions. Identifying a mutant that disrupts this intermediate before Rad51 removal unifies existing models of Rad54-mediated D-loop formation and extension.