We have analyzed for function in DNA repair a large panel of BRCA1 missense substitutions derived from a large number of cancer cases, and we found that when the pathogenic mutants were mapped onto the three-dimensional structure of the BRCA1 BRCT domain and of the BRCA1-BARD1 RING domains that they formed clusters. While many non-functional mutants could be explained by prior knowledge, two epitopes on the surface of the amino-terminal RING domain and one epitope on the surface of the carboxy-terminal BRCT domain have unknown function. In the case of the cluster on the BRCT domain, we hypothesize that it is a hitherto unknown protein binding site on the opposite face of the protein from the already known phosphoprotein binding site important for DNA repair. Similarly, the two epitopes in the amino-terminus of BRCA1 are likely protein-protein interaction sites with unknown binding partners, but we do know that these sites are critical for DNA repair function. We conclude that these cancer-associated mutations define novel protein binding sites that are important for BRCA1 function in DNA repair and for tumor suppression activity. This project will identify proteins that bind to BRCA1 dependent upon these epitopes. Since the mutation of these amino acids impairs BRCA1 function in DNA repair and tumor suppression, we anticipate that the proteins that bind to these amino acids will be tumor suppressors and DNA repair factors. In this early phase project, we will identify the proteins that bind to these epitopes by comparing proteins purified from wild-type versus specific mutant BRCA1 proteins. We will utilize quantitative mass spectrometry, which is the most sensitive method to detect such an interaction. Identified proteins will be analyzed/validated for function in DNA repair and will be analyzed in cancer databases for potential mutations in tumors. This project will identify one, or several, proteins that bind to each epitope, validate them for DNA repair function, and will enable a larger study that delves into the potential tumor suppressor function of the identified proteins and further characterizes the DNA repair function initially identified as an outcome of this project.
Many proteins associated with cancer have yet to be discovered. From analyzing a large number of DNA changes that cause cancer, we have identified a part of the breast cancer associated tumor suppressor protein, BRCA1, that is associated with cancer and a specific DNA repair function in cells. In this project, we will use this revealed cancer-associated domain to 1) identify the protein binding partner, 2) test that binding partner in DNA repair function, and 3) evaluate the likelihood that mutation of this newly identified protein is associated with cancer.
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| Schrock, M S; Batar, B; Lee, J et al. (2017) Wwox-Brca1 interaction: role in DNA repair pathway choice. Oncogene 36:2215-2227 |
| Lee, Cindy; Banerjee, Tapahsama; Gillespie, Jessica et al. (2015) Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks. Hum Mutat 36:1205-14 |
