Firrm functions as a multiprotein interaction platform that coordinates Mcm8–9–Hrob recruitment and Fignl1-mediated Rad51 unloading

Rad51 forms nucleoprotein filaments to catalyze homology search and strand exchange. The transition to recombination-associated DNA synthesis is accompanied by Rad51 unloading and recombination helicase recruitment. However, how these reactions are coordinated remains unclear. Using Xenopus egg extracts, we identify a mechanistic coupling between Rad51 unloading and recombination helicase recruitment. Proteomic analysis revealed that Rad51 filaments assembled on double-stranded DNA recruit a limited set of recombination regulators, including C1orf112/Firrm, Fignl1, Mcm8, Mcm9, and Hrob. Firrm forms a stoichiometric complex with Fignl1 and directly binds short acidic motifs in the intrinsically disordered regions of both Mcm9 and Hrob. The Firrm–Mcm9 interaction promotes recruitment of Mcm8–9 and Hrob to Rad51 filaments. Purified Mcm8–9 stimulates the ATPase activity of Fignl1 through Firrm in a DNA-dependent manner. Rad51 unloading from double-stranded DNA depends primarily on Fignl1 ATPase activity and is stimulated by Firrm–Mcm9 interaction. In human cells, FIRRM–FIGNL1 and MCM8–9–HROB act in the same genetic pathway to confer resistance to DNA interstrand crosslinks. These results support a model in which Rad51 filaments and Firrm–Fignl1 promote recruitment of Mcm8–9–Hrob, which, in turn, stimulates Fignl1 ATPase activity, thereby coupling recombination helicase recruitment with Rad51 unloading.