Hot Spot Analysis of the Breast Cancer Susceptibility Protein

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Abstract Germline mutations in the breast cancer susceptibility gene (BRCA1) are heavily linked to familial breast and ovarian cancers. Women who inherit these mutations are ~60% more likely to develop the disease. During normal female development, critical windows of vulnerability correlate with the early onset of breast cancer. These events coincide with a buildup of DNA lesions in mammary tissue as reactive oxygen species are formed from the metabolic processing of estrogen. Damaged DNA, if left unrepaired, can perpetuate errors in the genome. Reduced expression levels of the BRCA1 protein or BRCA1 mutations, compounded with insufficient lesion repair, provide a tipping point toward cancer induction. At the molecular level, the intricate details of these mechanisms are poorly understood. In the nucleus, BRCA1 associates with its binding partner the BRCA1-Associated Ring Domain protein (BARD1) to help coordinate the repair of DNA modifications. The BRCA1-BARD1 heterodimer performs these operations by interacting with other repair proteins, such as BRCA2, at damaged sites on DNA. In this context, BRCA1 acts as a tumor suppressor to ensure fidelity in the genome. Inherited mutations in BRCA1 can cause functional deficiencies in the protein that affect its job in tumor suppression. Moreover, as decades of scientific research demonstrate BRCA1's multifaceted role in DNA repair, information about the physical properties of BRCA1 are just coming to light. Improving our knowledge of BRCA1's three-dimensional (3D) structure can provide new insights for therapeutic discovery. In the proposed research, we will use a unique combination of cryo-Electron Microscopy (EM) imaging technology and biochemical tools to study the differences in wild type and mutated BRCA1. Our preliminary data suggests that changes in mutated BRCA1 in response to oxidative damage leads to changes in a “modification hot spot” on the protein. We will test this idea across multiple breast cancer cells lines, then develop a new enzymatic approach to attenuate this effect in cancer cells. Our combined biochemical and structural biology strategies will provide a new lens to view the physical nuances of the BRCA1 structure for the first time. This information will shed light on BRCA1 deficiencies relate to cancer susceptibility.