Mazin, A. | Search bioRxiv

bioRxiv2026

FBH1 and RAD54L directly interact and cooperate to drive replication fork reversal

Replication fork reversal alleviates DNA replication stress and maintains genome stability. We previously showed that FBH1 and RAD54L cooperate to promote fork reversal in human cells, and that FBH1-dependent fork reversal requires the branch migration activity of RAD54L. However, the molecular basis of this cooperation remained unclear. Here, we identify both a physical and functional interaction between FBH1 and RAD54L. We demonstrate that purified FBH1 and RAD54L interact directly and form a complex at stalled replication forks in cells. Mapping studies revealed that RAD54L Lobe 1 is critical for interaction with the FBH1 2B subdomain. In cells, FBH1-RAD54L complex formation is enhanced in the absence of RAD51. Consistently, purified RAD54L displays a stronger affinity for RAD51 than for FBH1. Using biochemical reconstitution assays, we further show that FBH1 and RAD54L promote fork reversal more efficiently together than either protein alone, with maximal reversal observed when FBH1 acts before RAD54L. Collectively, our findings establish RAD54L as an essential functional partner of FBH1 in replication fork reversal and provide mechanistic insight into the sequential coordination of their activities.

cell biology↗

bioRxiv2024

Distinct roles of the two BRCA2 DNA binding domains in DNA damage repair and replication fork preservation

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DNA binding domains in genome maintenance.

biochemistry↗