A New Cell-Free System to Study BRCA1 Function

Abstract

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.

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Document Details

Document Type
Technical Report
Publication Date
May 01, 2014
Accession Number
ADA608618

Entities

People

  • Betty Diamond
  • Johannes Walter

Organizations

  • Harvard Medical School

Tags

Communities of Interest

  • Biomedical

DTIC Thesaurus Topics

  • Antibodies
  • Biomedical Research
  • Biomolecules
  • Cell-Free System
  • Cells
  • Chemical Compounds
  • Chromosome Structures
  • Department Of Defense
  • Dissociation
  • Electronic Mail
  • Enzyme Inhibitors
  • Information Operations
  • Mass Spectrometry
  • Mutant Proteins
  • Proteins
  • Spectrometry
  • Unloading

Readers

  • Military Engineering.
  • Molecular Genetics
  • Molecular and genetic basis of cancer.