Distinct complexes of RAD51 paralogs participate in different fork protection pathways

Abstract Error-free genome duplication is critical for cellular homeostasis and genome maintenance. Stalled forks undergo remodeling during replication stress, and unprotected forks undergo degradation by nucleases. RAD51 paralogs are evolutionarily conserved essential proteins for genome maintenance with diverse roles ranging from homologous recombination (HR) to replication stress responses. However, the mechanisms underlying their participation in fork maintenance remain unclear. Here, we demonstrate that the RAD51 paralogs do not participate in the canonical SMARCAL1-BRCA2 axis of fork protection. Instead, RAD51D–XRCC2 (DX2) and RAD51C–XRCC3 (CX3) complexes protect forks remodeled by the FBH1 helicase, whereas the RAD51B–RAD51C (BC) subcomplex but not BRCA2 safeguards forks remodeled by the FANCM translocase, revealing a new FANCM-mediated pathway of fork remodeling which is protected by the BC sub-complex. Mechanistically, we show that FANCM-mediated fork reversal is RAD51-dependent and generates substrates for MRE11-, EXO1-, and DNA2-mediated degradation in the absence of RAD51B/C. Our findings establish the participation of the RAD51 paralogs in multiple fork protection pathways in a fork-remodeler-specific manner, highlighting the existence of several independent fork remodeling and protection mechanisms for maintaining genomic stability under replication stress.