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A New Cell-Free System to Study BRCA1 Function

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Annual rept. 1 May 2014-30 Apr 2015

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This proposal is based on our finding that in a cell-free system based on Xenopus egg extracts, the tumor suppressor BRCA1 is required for a novel step in the repair of DNA interstrand cross-links ICL. Specifically, prior to our application of funding, we had found that in the absence of BRCA1, when replication forks collide with an ICL, leading strands stall 20 nucleotides from the ICL and fail to be extended towards the ICL lesion. So far, we have shown that leading strand extension is critical for ICL repair. In addition, we found that in BRCA1-depleted egg extracts, the CMG helicase that unwinds DNA ahead of DNA polymerases, fails to be unloaded from the stalled fork. This explains the leading strand arrest at the -20 position and identifies a potentially new function for BRCA1 in ICL repair and tumor suppression. We have also developed new ways of inhibiting BRCA1 function in egg extracts and examined the role of potential BRCA1 effectors FANCJ, FANCM, CTIP in promoting the extension step. We conclude that BRCA1 does not perform its function by acting through FANCJ, FANCM, or CTIP. In addition, we have shown that BRCA1 is not regulated by whether one or two forks collide with the ICL. We also implicated the ATPase p97 in BRCA1-dependent CMG removal, we have identified ubiquitylated species of MCM7 that are likely involved in CMG unloading, and we have succeeded in expressing recombinant MCM2-7 complex in insect cells.

Subject Categories:

  • Biochemistry
  • Genetic Engineering and Molecular Biology
  • Anatomy and Physiology
  • Medicine and Medical Research

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