Probing the BRC repeats RAD51 interaction by integrating computational and experimental biophysics

The BRCA2/RAD51 interaction is a critical step of homologous recombination, a key mechanism for maintaining genome integrity. BRCA2 recruits and transports RAD51 to DNA damage sites through eight short BRC repeats. Nowadays, only the interaction of RAD51 with BRC4, the fourth repeat, has been structurally characterized, leaving the role of the other BRC repeats unclear. To shed light on the close relationship between these two proteins we are combining experimental and computational approaches. We first used AlphaFold simulations to predict peptide binding modes and rank their affinities. To further rationalize these insights, we performed residue scanning analyses and molecular dynamics (MD) simulations. We then isolated a human monomeric RAD51 form to study the interaction of individual BRC repeat peptides using orthogonal biophysical experiments. The calculated affinities were correlated with the ability of isolated BRC repeats to interact with RAD51 WT, revealing that only the highest affinity peptides could disassemble RAD51 fibrils. These findings guided the design of RAD51 complexes with the strongest binders, either alone or within BRCA2 truncations containing multiple peptides, which were subsequently used for structural investigations. Our results, obtained by integrating AlphaFold predictions with XL-MS, NMR and SAXS data, unveil not only the crucial role of BRC repeats interaction with RAD51’s N-terminal domains, but also on the dynamic and multifaceted behavior of the regions linking the first four BRC repeats. This suggests that these peptides may cooperate to disassemble RAD51 fibrils, thus allowing RAD51 translocation into the nucleus. As a future perspective, we would like to integrate information gathered through other structural techniques with CryoEM to provide novel insights into the interaction of these complexes with DNA.