RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

Fanconi anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. Mutations in RAD51 paralogs have been identified in FA-like disorders and cancers. Although the role of RAD51 paralogs is well established in homologous recombination (HR)-mediated DNA repair, little is known about their role during replication stress responses. Here, we report that the RAD51C-XRCC3 (CX3) complex of RAD51 paralogs participates in the FA-pathway of R-loop tolerance mechanism. CX3 complex suppresses R-loops, transcription-replication collisions (TRC), and associated genome instability in physiological and replication stress conditions. Mechanistically, the CX3 complex physically interacts with FANCM and facilitates its recruitment to the R-loop sites to promote its resolution. Notably, cells expressing RAD51C R258H pathological mutant exhibit defective interaction with FANCM and display inefficient R-loop processing. Strikingly, the R-loop resolution function of RAD51C is independent of its ATPase activity, which is required for HR, fork protection, and restart. Collectively, our work identifies a novel role of the CX3 complex in preventing R-loop-induced genome instability by regulating FANCM-mediated R-loop resolution.