Integration of computational and experimental biophysics reveals novel insights on the BRCA2 - RAD51 interaction

The interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination pathway, is a key-process for the integrity of our genome. Through eight short repeats, BRCA2 recruits and transports RAD51 to the sites where DNA damage is processed. Nowadays, only the interaction of RAD51 with BRC4 has been structurally characterized, through X-ray crystallography, by removing the first 97 amino acids of RAD51. Nevertheless, very little biophysical data and no structural information are available on the interaction of the other BRC repeats with RAD51. To shed light on the tight relation between these two proteins we decided to combine experimental and computational approaches. So far, the structural complexity and dynamics of RAD51 have severely hampered our understanding of these interactions. Therefore, we isolated a novel fully human monomeric RAD51 form which was exploited to study the interaction of isolated BRC repeats peptides through orthogonal biophysical experiments. The calculated affinities were then correlated to the ability of the isolated BRC repeats to interact with RAD51 WT, revealing that only peptides with the highest affinities could disassemble RAD51 fibrils. To further rationalize the peptides’ affinities, we performed residue scanning analyses and molecular dynamics (MD) simulations. In particular, the former was applied to estimate the per-residue relative change in binding affinity for each BRC repeat, while MD simulations were used to observe the conformational dynamics associated with the different RAD51-BRC repeat complexes. Interestingly, these results revealed that specific amino-acid variations of the BRC repeats affect their binding to RAD51, and specifically to its N-terminal domain. To support these data and get further insights into the interactions of the full length RAD51 with the BRC repeats, we are aiming to integrate Alphafold2 predictions, SAXS data and MD simulations. As a future perspective we would like to apply this approach, in combination with Cryo-EM, to study the conformational dynamics of BRCA2 truncates containing multiple repeats (e.g. BRC3-4) in complex with RAD51 to unravel novel insights on the role of the spacing regions that separate different BRC repeats.