RAD51 Paralogs and RAD51 Paralog Complexes BCDX2 and CX3 Interact with BRCA2

Abstract

Homologous recombination (HR) is an important mechanism for repairing DNA double-strand breaks (DSBs) and preserving genome integrity. Pathogenic mutations in the HR proteins BRCA2 and the RAD51 paralogs predispose individuals to breast, ovarian, pancreatic, and prostate cancer. The RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 form two complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3). Similar to BRCA2, loss of RAD51 paralog functions in mammalian cells lead to chromosomal abnormalities, growth defects, disrupted RAD51 foci formation, and PARP inhibitor sensitivity. Despite significant e]ort over the past three decades, the specific molecular functions of the human RAD51 paralogs have remained elusive due to technical challenges such as low protein expression in human cell lines and instability of the purified proteins. Recent studies have determined the molecular structures of the BCDX2 and CX3 complexes dramatically enhancing our understanding of these challenging proteins. Using multiple approaches, we demonstrate that the RAD51 paralogs interact with BRCA2 at two distinct interaction hubs located in the BRC repeats and the DNA binding domain. We confirm, using a yeast 3-hybrid approach, that human RAD51 paralogs interact directly with BRC repeats one and two (BRC1-2) of BRCA2. Because of the dynamic nature of the RAD51B C-terminal domain (CTD), identified in the recently solved cryo-EM structures, we focused on elucidating the interaction with RAD51B. We determined that BRCA2 interacts with the CTD of RAD51B and not the N-terminal domain (NTD) that is involved in stacking interactions with RAD51C and RAD51D. Furthermore, the interaction with RAD51B is dependent upon an FxxA motif located on a surface exposed region of the CTD. Our study has identified novel interactions between the RAD51 paralogs and BRCA2 and further demonstrated that a previously unrecognized FxxA motif located within a mobile element of RAD51B is critical for the interaction.