An integrated biophysical and structural biology approach to investigate the BRCA2-RAD51 interaction

The interaction of RAD51 and BRCA2 is a central process in homologous recombination (HR), a DNA repair pathway that preserves genome integrity by fixing double strand breaks. BRCA2 recruits, transports and assist RAD51 at damaged DNA sites through eight conserved BRC repeats. To date, only the interaction between RAD51 and the fourth BRC repeat (BRC4), has been structurally characterized, while the role of the other BRC sequences remain unclear. Nevertheless, the ability of RAD51 to interact with multiple BRC repeats suggest the existence of a finely tuned regulatory mechanism controlling RAD51 activity. The main goal of this work is to elucidate mechanistic details of RAD51BRCA2 interaction and clarify the tight correlation between these proteins, their role in genome stability and cancer susceptibility through an integrated biophysical and structural approach. Firstly, the interaction of isolated BRC repeats with RAD51 was thoroughly characterized within our group, using complementary biophysical experiments and a novel fully human monomeric RAD51. The measured binding affinities were correlated with the ability of the individual BRC repeats to disrupt RAD51 WT fibrils, revealing that only high affinity peptides effectively induce filament disassembly. To obtain further insights into the interactions between the fulllength RAD51 and individual BRC repeats, SAXS, XLMS experiments were integrated with MD simulations. Finally, BRCA2 truncates containing multiple repeats (e.g. BRC34, BRC14) were successfully coexpressed and copurified in complex with RAD51. Ongoing structural investigations combining CryoEM, XLMS, SAXS and computational approaches aim to unveil the architecture and dynamic behavior of these complexes. The integration of these results is expected to provide mechanistic explanations for diseaserelated mutations affecting RAD51BRCA2 binding and to support the development of personalized therapeutic strategies involving the DNA repair pathways.