Pathogenic BRCA1 DBD variants exhibit altered DNA binding affinities and susceptibility to menadione

Abstract

Breast Cancer Susceptibility Gene 1 (BRCA1) codes for a DNA repair protein that facilitates the repair of double-stranded DNA breaks (DSBs) in human cells through the homologous recombination (HR) pathway. Mutations of BRCA1 are highly associated with breast cancer; however, many variants remain unclassified with unknown cellular phenotypes. The DNA binding activity of BRCA1 is localized primarily to its central region, which can be divided into two distinct domains: DNA Binding Domain 1 (DBD1; amino acids (aa) 330-554) and 2 (DBD2; aa 894-1057). We previously proposed a model in which DBD1 targets BRCA1 to DSBs for the promotion of DNA end resection, while DBD2 targets BRCA1 to telomeres to function in chromatin remodeling and telomere regulation. In this study, we hypothesized that unknown DBD variants (T374I, K408E, N417S, N909I, M1008I, and R1028H) with similar properties to known disease-causing variants (Q356H, F461L, R496H, D940Y, S1027N, and E1038G) would also be pathogenic. The affinities of each variant for single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and a G-quadruplex (G4) sequence were measured via biolayer interferometry. The DNA repair phenotypes of each variant were analyzed by overexpression in HEK cells to determine correlation between binding activity and DNA damage response. Altogether, these results provide insight into how missense mutations affect the ability of BRCA1 DBDs to facilitate the DNA damage response.