Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage

Mala Mani, Jeff Smith, Karthikeyan Kandavelou, Jeremy M. Berg, Srinivasan Chandrasegaran

Research output: Contribution to journalArticlepeer-review

Abstract

Custom-designed zinc finger nucleases (ZFNs) are becoming powerful tools in gene targeting-the process of replacing a gene within a genome by homologous recombination. Here, we have studied the DNA cleavage by one such ZFN, ΔQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by ΔQNK-FN is greatly facilitated when two ΔQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of ΔQNK-FN, indicating that double-strand cleavage requires dimerization of the FokI cleavage domain. Rates of DNA cleavage decrease as the substrate concentrations increase, suggesting that the ΔQNK-FN molecules are effectively "trapped" in a 1:1 complex on DNA when the DNA is in excess. The physical association of two ZFN monomers on DNA was monitored by using the biotin-pull-down assay, which showed that the formation of ΔQNK-FN active complex required both binding of the two ΔQNK-FN molecules to specific DNA sites and divalent metal ions.

Original languageEnglish (US)
Pages (from-to)1191-1197
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume334
Issue number4
DOIs
StatePublished - Sep 9 2005

Keywords

  • Chimeric nucleases
  • Directed mutagenesis
  • Gene targeting
  • Gene therapy
  • Homologous recombination
  • Homology-directed repair
  • Non-homologous end joining
  • Protein engineering
  • Sequence-specific cleavage
  • Zinc finger nucleases

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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