An integrative structural biology study of the RAD51–BRC repeats interplay in Homologous Recombination

Homologous recombination (HR) is a critical repair pathway involving numerous proteins that ensure error-free DNA double-strand breaks (DSBs) repair. Despite advances, many aspects of HR remain poorly understood. Notably, even one of the earliest identified and most critical interactions, between RAD51 and BRCA2, remains incompletely characterized, mainly due to the lack of structural data. BRCA2 interacts with RAD51 through eight short repeats, and through an additional C-terminal BRCA2 domain. Nowadays, only the interaction of RAD51 with the fourth BRC repeat, has been structurally characterized while the roles of the remaining BRC sequences are not yet fully defined. Indirect biochemical evidence suggests that the first four repeats may inhibit DNA binding, whereas BRC5-8 have the opposite effect, promoting RAD51-DNA interaction. This points to distinct roles for different repeats during RAD51 nucleation on resected ssDNA. Nevertheless, direct biophysical data on the individual contributions of the BRC repeats to RAD51 nuclear recruitment, a crucial step in HR, are still lacking. Such information would be invaluable for guiding the rational design of novel anticancer therapies targeting this critical interface, which is already recognized as a validated target in “chemically induced synthetic lethality” strategies. To shed light on the molecular relationship between these two proteins, we combined computational methods, biophysical experiments, and integrative structural biology. We aimed to comprehensively characterize the interaction of each BRC repeat with RAD51 and to unveil novel structural insights into both isolated BRC-repeats and larger BRCA2 truncation constructs encompassing multiple peptides and their linker regions. Beginning with an assessment of the correlation between the binding affinities of individual BRC repeats and their impact on RAD51 disassembly, our investigation extends to larger BRCA2 truncations, offering unprecedented insights into the molecular determinants of RAD51 recognition.