| Description |
Although BRCA1 has multiple roles in the cell, its activity in homology-directed DNA repair (HDR) is most closely associated with cancer risk. BRCA1, which encodes a protein of 1,863 amino acids, harbors two highly conserved domains: an amino-terminal RING domain and two carboxy-terminal BRCT repeats. All of the known pathogenic missense substitutions in this protein occur in these two domains, unless the underlying nucleotide change is spliceogenic. While mutations in both of these domains are associated with increased cancer risk, it is still unclear how non-spliceogenic RING missense substitutions elicit their pathogenicity. Two well-characterized functions of the RING domain are its interaction with BARD1 and E3 ubiquitin ligase activity. Records linking full-sequence tests of BRCA1 to previously collected high-throughput functional data measuring these two activities showed only an enrichment of single-nucleotide substitutions at BRCA1 residues exclusively associated with heterodimerization. This is in agreement with recent structural and biochemical studies that put heterodimerization upstream of E3 ubiquitin ligase activity. These results, and concerns about the accuracy and indirect nature of the aforementioned functional assays, guided our development of a one-by-one mammalian two-hybrid assay. Subsequent calibration of this assay led to the classification of one newly reported BRCA1 missense substitution (BRCA1 p.P34S). Moving forward, we envision converting this assay into a high-throughput format to obtain BARD1-binding iv scores for all possible BRCA1 RING missense substitutions that can be reached by a single-nucleotide change. |
