Dissecting the BRCA2 - RAD51 interaction by integrating computational and experimental biophysics

The interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination pathway for the repair of DNA double-strand breaks (DSB), is a key-process for the integrity of our genome. Alterations of their interaction, which have been associated with cancer development, lead to defects in the DNA DSB repair pathway. Through eight short repeats, BRCA2 recruits and transports RAD51 to the sites where DNA damages are processed. Several literature reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with RAD51. Nowadays, only the interaction of RAD51 with the fourth BRC repeat 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. My research project aims at elucidating the tight relation between these two proteins by combining experimental and computational approaches. Initially, the interaction of isolated BRC repeats with RAD51 was thoroughly characterized through orthogonal biophysical experiments exploiting a novel fully human monomeric RAD51, that was isolated in our laboratory. 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, using the Schrödinger suite of software. These computational approaches were used in a complementary spirit, as they provide static and dynamic information, respectively. 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 hRAD51, 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 planning to integrate SAXS, cross-linking mass spectrometry (XL-MS) experiments, and MD simulations. Moreover, we were able to co-express and co-purify BRCA2 truncates containing multiple repeats (e.g. BRC3-4) in complex with RAD51. We plan to perform structural investigations on this complex through Cryo-EM, SAXS and computational approaches to further investigate this interaction.