Abstract
BRCA1 (breast cancer-associated protein 1) plays a central role in homologous recombination (HR) through interactions with multiple proteins across its various domains. The C-terminal BRCT domains bind HR regulators such as ABRAXAS1, CtIP, and BRIP1, each contributing to distinct, sometimes opposing, functions. While pathogenic mutations frequently cluster within the canonical BRCT phospho-binding pocket, the broader mutational landscape and its functional consequences remain poorly understood. Here, we used a site-saturation mutagenesis library of the BRCT domains to test >4,000 single-residue variants for their ability to bind ABRAXAS1 and CtIP. Using a yeast two-hybrid screen, we systematically assessed these interactions and validated key findings in mammalian cells. The resulting interaction map identified previously uncharacterized residues critical for partner binding and demonstrated their detrimental impact on HR. Importantly, we identified separation-of-function mutations that selectively disrupt individual protein interactions, enabling a more detailed analysis of each partner's contribution to HR. Functional assays suggested that disruption of CtIP binding had the most pronounced impact on HR. Furthermore, integration of our data with clinical variant data revealed a strong correlation between loss of protein binding and pathogenicity, highlighting the potential utility of our interaction map for clinical variant interpretation.