Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination

Derek J.R. Ransburgh, Natsuko Chiba, Chikashi Ishioka, Amanda Ewart Toland, Jeffrey D. Parvin

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

Effects of breast cancer-associated gene 1 (BRCA1) missense mutations on the function of BRCA1 protein in DNA recombination have been little studied. In this report, we adapted a homology-directed recombination (HDR) assay to analyze the effects of BRCA1 mutations on this function. Using a HeLa-derived cell line with a genomically integrated recombination substrate, we expressed an endonuclease creating a double-stranded break in the substrate that the HDR assay scores by generation of green fluorescent protein-positive cells. By combining RNA interference (RNAi) that targets cellular BRCA1 mRNA with expression of RNAi-resistant BRCA1 mutants, we could effectively substitute selected point mutants to test these in the cellular recombination assay. We found that ∼300 residues at both termini of the BRCA1 protein were essential for HDR. Whereas some mutations analyzed were neutral, mutations that altered any zinc-coordinating residue or generated M18T and T37R alterations were defective for recombination. This study established a robust assay system to analyze the function of BRCA1 in regulating homologous recombination, which is critical for its tumor suppressor function.

Original languageEnglish
Pages (from-to)988-995
Number of pages8
JournalCancer Research
Volume70
Issue number3
DOIs
Publication statusPublished - 2010 Feb 1

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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