A zinc finger nuclease assay to rapidly quantitate homologous recombination proficiency in human cell lines

Anal Biochem. 2013 Mar 1;434(1):96-8. doi: 10.1016/j.ab.2012.11.002. Epub 2012 Nov 10.

Abstract

Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.

MeSH terms

  • BRCA1 Protein / antagonists & inhibitors
  • BRCA1 Protein / genetics
  • BRCA1 Protein / metabolism
  • BRCA2 Protein / genetics
  • BRCA2 Protein / metabolism
  • Cell Line, Tumor
  • Endonucleases / metabolism*
  • Enzyme Assays*
  • HeLa Cells
  • Homologous Recombination*
  • Humans
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Zinc Fingers

Substances

  • BRCA1 Protein
  • BRCA2 Protein
  • RNA, Small Interfering
  • Endonucleases