Francisco E. Neal1,2 ∙ Wenjing Li1 ∙ Mollie E. Uhrig3,4 ∙ Jeffrey N. Katz1,2 ∙ Shahrez Syed1,2 ∙ Neelam Sharma3 ∙ Arijit Dutta1,2,8 ∙ Sandeep Burma1,5 ∙ Robert Hromas6 ∙ Alexander V. Mazin1 ∙ Eloise Dray1 ∙ David S. Libich1,2 ∙ Shaun K. Olsen1,2 ∙ Elizabeth V. Wasmuth1,2 ∙ Weixing Zhao1,2 ∙ Claus S. Sørensen7
Highlights
BRCA2 OB-fold DNA-binding domain has specificity for ssDNA
BRCA2 C-terminal DNA-binding domain has specificity for dsDNA
OB-fold DNA-binding domain is needed for DNA repair and replication fork protection
C-terminal DNA-binding domain functions primarily in replication fork protection
Summary
Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, 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 DBDs in genome maintenance.