Control of Commitment Steps in Mammalian Homologous Recombination

Project Details

Description

Project Summary: ?Control of Commitment Steps in Mammalian Homologous Recombination? The appearance of many cancer cases, especially breast, ovarian, and pancreatic cancer, is linked to problems with DNA repair. Deficiencies in key DNA repair genes, such as BRCA1, PALB2 and BRCA2 , are associated with inefficient DNA repair by the homologous recombination (HR) pathway. Each of these factors is required to load the key HR intermediate, RAD51, at DNA double-strand break sites. The exact mechanism by which BRCA1 is recruited to DNA break sites and becomes activated at those break sites is not fully understood. HR efficiency is also affected by the activity of certain regulators. 53BP1 regulates HR by reducing BRCA1-mediated loading of RAD51. RNF4 is reported to regulate recruitment of BRCA1 to DNA breaks, and increased abundance of RNF4 correlates with poor survival in breast cancer. The overall goal of this proposal is to clarify how BRCA1 becomes localized and activated at DNA breaks, and how interactions between BRCA1 and PALB2 mediate productive HR. This goal will be realized by investigating three mechanisms that control recruitment of the BRCA1-associated repair machinery. First, we will test how post-translational modification of proteins at break sites by ubiquitination and SUMOylation affects BRCA1-mediated HR. In particular, we will focus on RNF4, a ?SUMO-targeted E3 ubiquitin ligase? with a known role in DNA repair, which appears to affect the growth of cancer cells. Using a conditional-knockout mouse model that was recently generated by our lab, we will test how RNF4-mediated ubiquitination affects turnover of repair components at DNA breaks, and how this affects cell survival after DNA damage. Secondly, we will test the contributions of two factors that appear to be important for recruitment of BRCA1 to DNA damage sites. These factors are BARD1, a RING domain-containing protein that interacts constitutively with BRCA1; and TOPBP1, a large scaffold protein that loads onto chromatin at DNA damage sites and recruits multiple DNA repair factors. Data obtained in our lab using genetically-modified mouse models and mass spectrometry supports a role for BARD1 and TOPBP1 in BRCA1 recruitment, and this project will address how important they are for BRCA1-mediated HR. The third part of the proposal focuses on interactions between BRCA1 and PALB2. BRCA1 is able to form a complex with PALB2 by association of helical elements in both proteins to form a ?coiled coil? domain. The heterodimer of BRCA1:PALB2 stabilizes BRCA2 at the break site, which in turn helps load RAD51 to facilitate HR. PALB2 can also form a coiled coil by homodimerization, however, which prevents it from interacting with BRCA1 and limits the efficiency of HR. The ability of PALB2 to form non-recombinogenic and pro-recombinogenic complexes is proposed to regulate HR efficiency, hence we have studied the different complexes in detail using NMR spectroscopy. By studying and testing our NMR structures of PALB2 complexes, we will dissect the contributions of these different oligomeric forms to regulation of HR. This research will deepen our understanding of regulation of repair, and inform future strategies to prevent mutation caused by defective DNA repair.
StatusActive
Effective start/end date9/1/151/31/22

Funding

  • National Cancer Institute: $351,543.00
  • National Cancer Institute: $340,996.00
  • National Cancer Institute: $351,543.00
  • National Cancer Institute: $74,539.00
  • National Cancer Institute: $351,543.00
  • National Cancer Institute: $351,543.00
  • National Cancer Institute: $319,991.00

ASJC

  • Genetics
  • Molecular Biology

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