Elucidating the BRCA2-RAD51 interaction through an integrated biophysical and structural biology approach

The interaction of RAD51 and BRCA2, two proteins involved in the homologous recombination (HR) pathway for the repair of DNA double strand breaks (DSB) is a key-process for the integrity of our genome (1). BRCA2 recruits, transports and assist RAD51 to the sites where DNA damages are processed (2). BRCA2 interacts with RAD51 through eight short repeats with different affinities, and through an additional C-terminal BRCA2 domain (1). Nowadays, only the interaction of RAD51 with the fourth BRC repeat, has been structurally characterized while it is not completely clear the role of other BRC sequences (3, 4). Nevertheless, the interaction of RAD51 with multiple BRC repeats may imply a tight and fine regulation of RAD51 activity. Several reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity (5). 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. To shed light on the close relationship between these two proteins we combined 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 integrative structural investigation, encompassing cross-linking mass spectrometry, small angle X-ray scattering and AlphaFold simulations. Our results shed light not only on 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.