The mechanistic understanding of the BRCA2-RAD51 interaction: an integrated structural biophysics study

The interaction of RAD51 with BRCA2 is a critical step of homologous recombination, a mechanism that maintains genome integrity. 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, due to the structural complexity and dynamics of RAD51, the mechanistic details of each step of RAD51 recruitment and DNA repair still need to be elucidated. We performed an integrative experimental-computational approach to shed light on the tight relation between these two proteins. We evaluated the mechanism of RAD51 defibrillation driven by BRC4 through negative staining transmission electron microscopy (TEM) experiments and size exclusion chromatography, revealing that BRC4 erodes RAD51 fibrils from their termini. Nevertheless, the propensity to oligomerization of the WT protein limited further biophysical studies. Therefore, we isolated a novel, fully human monomeric RAD51 form, which was exploited to study the interaction with BRC4 through orthogonal biophysical methods. SAXS experiments were also carried out on the full-length monomeric protein and its complex with BRC4 peptide, providing novel structural insights into their behavior in solution. Specifically, atomistic modeling of AlphaFold2 predictions revealed that both proteins display flexible N-terminal domains. These results and previous evidence on RAD51 WT fibrils suggest that BRC4 binding triggers a conformational rearrangement of the RAD51 N-terminal domain from a more ordered to an intrinsically disordered state. These data allowed a thorough understanding of the interaction between BRC4 and RAD51, suggesting that disassembling the RAD51 fibril operated by BRCA2 in the cytosol is the first critical step of homologous recombination. Importantly, we proved that the isolated RAD51 monomeric form is a novel and reliable platform for structural and biophysical analyses of the BRC repeats-RAD51 interaction. Indeed, this tool will help future works aimed not only at elucidating the role of the other BRC repeats in the cytosolic recruitment of RAD51 but also to understand how they support the priming of RAD51 to the site of DNA damage. Finally, the BRCA2-RAD51 interaction has been reported as an appealing target for developing anti-cancer treatments since its disruption would mimic a BRCAness condition. In this context, the concomitant administration of PARP inhibitors synergistically results in cell death, enlarging the synthetic lethality paradigm. Therefore, further understanding the BRCA2-RAD51 interaction will provide essential information for innovative anti-cancer therapies.