Although germline mutations of the BRCA1 gene are a major cause of familial breast cancer, the molecular mechanisms by which BRCA1 suppresses tumorigenesis are still poorly understood. My dissertation project is to analyze the biological function of the ring domain and the BRCT domain of BRCA1. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein and has potent E3 ligase activity within its ring domains. Ubiquitylation is a stepwise process involving E1, E2 and E3 enzymes that mediate the covalent attachment of ubiquitin to a target protein. The E3 ubiquitin ligase catalyzes the transfer of an ubiquitin molecule from the E2 enzyme to the substrate. BRCA1 also has two BRCT repeats that have phosphopeptide binding activity. Studies indicate that this domain is important for the role BRCA1 plays in DNA repair and transcriptional regulation.
SPECIFIC AIMS : To determine whether the E3 ubiquitin ligase and phosphopeptide binding activity of BRCA1 is required for 1) normal animal development, 2) BRCA1- mediated mammary tumor suppression, or 3) Brcal function in DNA double strand break repair. Study Design &Methods: To address my first aim, I am using a knock-in gene targeting approach to generate mice with specific point mutations within the BRCA1 gene that eliminate either the E3 ubiquitin ligase activity or the phosphopeptide binding activity. The phenotype of these Brcal mutant mice will then be compared to those of wild type and other existing Brcal mutant mouse models. To address my second aim, a cre/lox gene targeting system will be used to generate mice strains that express, in a mammary-specific fashion, the BRCA1 mutant alleles. If the E3 ubiquitin ligase or phosphopeptide binding activity is required for mammary tumor suppression, then these mice should develop breast carcinomas with a latency and histopathology similar to mice that harbor mammary-specific Brcal- or Bard1-null mutations. To test the third hypothesis, I will establish cell lines that only express the functionally-inactive BRCA mutant alleles and will determine whether these activities are required for double strand DNA break repair and the various cell cycle checkpoints in which BRCA1 has been implicated. These finding are important in terms of public health because cancer is the second leading cause of death and specifically breast cancer is the second leading cause of death for women in the United States. Many cancer predisposing mutations have been identified within the ring domain and BRCT repeats of BRCA1 in human breast cancer cases. These findings could also lead to more specialized treatments and overall improved health care for cancer patients, which supports the mission of the National Cancer Institute.
|Shakya, Reena; Reid, Latarsha J; Reczek, Colleen R et al. (2011) BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity. Science 334:525-8|
|Reid, Latarsha J; Shakya, Reena; Modi, Ami P et al. (2008) E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks. Proc Natl Acad Sci U S A 105:20876-81|