Postdoc, Yale Medical School
BRCA2 DNA repair and Breast and Ovarian Cancer progression
Ovarian cancer is the fifth leading cause of cancer death among U.S women accounting for more deaths than any other cancer of the female reproductive system, and about 5% of women in US will develop lethal ovarian cancer during their lifetime. BRCA2 (Breast Cancer Susceptibility Gene 2) was originally identified as one of two genes responsible for familial inherited breast and ovarian cancer. Women who inherit a mutant copy of BRCA2 have up to an 80% lifetime risk of developing breast cancer. BRCA2 is directly involved in a cellular process termed DNA repair to maintain genome integrity. Malfunctions in DNA repair proteins, which are quality control inspectors, can lead to a process termed “genomic instability” whereby mutations throughout the genome are accumulated at an accelerated rate. If these mutations lie in specific genes that control cellular division or death, a cell may acquire the capability to seed a tumor. The BRCA2 protein protects against this process by repairing damaged DNA and suppressing the accumulation of mutations through a specialized process called homologous recombination. The clinical options for preventing future cancers in BRCA2 mutation carriers involve prophylactic bilateral mastectomy and salpingo-oophorectomy, major surgical interventions that may be emotionally difficult for patients. The recent advent of PARP inhibitors as a synthetic lethal strategy to target BRCA mutant tumors while promising, may not have the efficacy that was initially expected and resistance to this therapy is already emerging in some patients. We still lack a basic, molecular understanding of how breast tumors are initiated in BRCA2 carriers which can help to early diagnosis, as well as how tumors acquire resistance to our current standard-of-care treatments. Elucidation of the underlying BRCA2 biology may unveil pathways or genes that could lead to initial prevention strategies targeting the early steps of tumor formation for these patients. Therefore, my main objective in this study is to dissect the molecular changes that drive ovarian tumor initiation in the setting of BRCA2 dysfunction. In order to address this issue, we have created a human fallopian tube cell system where we can regulate the levels of BRCA2 mimicking the acute loss of BRCA2 function that occurs in BRCA2 carriers. We have validated that our system effectively controls the levels of BRCA2 and we have confirmed the hallmarks of BRCA2 loss in fallopian tube cells.
Abstract: The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
Pub.: 07 Jul '15, Pinned: 20 Jul '17
Abstract: BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5-8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1-4. Furthermore, BRC5-8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5-8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.
Pub.: 17 Apr '16, Pinned: 20 Jul '17