Domain-Specific DNA Binding Activities of BRCA1 Reveal Substrate Preferences for Homologous Recombination and Telomere Regulation

BRCA1 is a crucial component of homologous recombination (HR), a high-fidelity pathway for repairing double-stranded DNA breaks (DSBs) in human cells. The central region of the BRCA1 protein contains two putative DNA binding domains (DBDs), yet their relative substrate specificities and functional contributions to HR remain unclear. Here, we characterized the DNA binding properties of DBD1 (amino acids 330-554), DBD2 (amino acids 894-1057), and BRCA1 C-terminal (BRCT) repeats using biolayer interferometry. Affinities were determined for single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex (G4) DNA. DBD2 exhibited strong and nearly identical binding to all three substrates (<i>K</i>_d = ∼35-44 nM), while the BRCT also bound to each structure similarly, but with lower affinity (<i>K</i>_d = ∼149-184 nM). In contrast, DBD1 showed a distinct preference for dsDNA, binding approximately 2-fold tighter compared to ssDNA or G4. These findings support a model in which BRCA1 uses modular DNA binding domains to recognize diverse repair targets; DBD2 serves as a primary anchor to associate with a broad range of DNA structures with BRCT contributing to the contacts. DBD1 acts as the determinant of DNA structure-specific localization that may help direct BRCA1 to DSB sites during HR or to noncanonical elements such as chromatin and telomeres. These insights lay the groundwork for future studies examining how cancer-associated variants affect the DNA binding and repair phenotypes of BRCA1 and may inform the interpretation of variants of unknown clinical significance.