A New Cell-Free System to Study BRCA1 Function
Annual rept. 1 May 2013-30 Apr 2014
HARVARD MEDICAL SCHOOL BOSTON MA
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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. In the last year, we have shown that leading strand extension is critical for ICL repair Aim 1. 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 Aim 2. 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.
- Medicine and Medical Research