A variety of DNA lesions lead to the formation of DNA double-strand breaks (DSBs), either directly or as intermediates of repair. To counter the accumulation of DNA damage, eukaryotic cells employ a complicated network of pathways that promote damage recognition, checkpoint signaling, and DNA repair. Components of the DNA damage response network have been linked to various genetic disorders that are typified by hypersensitivity to DNA damaging agents and cancer predisposition. In particular, the breast cancer tumor suppressor BRCA1 has been described as a master regulator of genome stability due to its involvement in various aspects of the damage response. This proposal seeks to understand how BRCA1 regulates homologous recombination (HR) to promote error-free repair of DSBs. In S phase, the ends of a DSB are processed by the resection machinery to promote HR-mediated repair, which takes advantage of the newly replicated sister chromatid as a template for error-free repair. Having recently established that Xenopus egg extracts can recapitulate recombination-dependent repair of a DSB, this system will provide a powerful tool to elucidate the mechanism of BRCA1-mediated HR. To study the dynamic events of HR, a novel DSB repair system will be established that supports analysis by single-molecule imaging. New techniques have been developed that support real-time imaging in highly concentrated Xenopus egg extracts, providing a significant advantage over traditional single-molecule approaches that rely on purified components studied in isolation. Single-molecule imaging will be used to analyze BRCA1-dependent DSB repair in real time, providing a level of mechanistic insight not available with traditional ensemble approaches. In this way, the complex functions of BRCA1 can be dissected to determine how cells regulate different aspects of the DNA damage response.

Public Health Relevance

BRCA1 is a well-established tumor suppressor gene that is frequently mutated in familial breast and ovarian cancers. BRCA1 plays a central role in regulating the cellular response to DNA damage and is critical to promote error-free repair and maintain genome stability. Understanding the mechanism of BRCA1-mediated repair will provide important insights into cancer development and help to identify additional treatment strategies for patients.

National Institute of Health (NIH)
Career Transition Award (K99)
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Special Emphasis Panel (ZGM1)
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Hamlet, Michelle R
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Harvard Medical School
Schools of Medicine
United States
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Klein Douwel, Daisy; Boonen, Rick A C M; Long, David T et al. (2014) XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol Cell 54:460-71
Long, David T; Joukov, Vladimir; Budzowska, Magda et al. (2014) BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork. Mol Cell 56:174-85