Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10769031 | Biochemical and Biophysical Research Communications | 2005 | 7 Pages |
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, ÎQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by ÎQNK-FN is greatly facilitated when two ÎQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of ÎQNK-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 ÎQNK-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 ÎQNK-FN active complex required both binding of the two ÎQNK-FN molecules to specific DNA sites and divalent metal ions.
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Authors
Mala Mani, Jeff Smith, Karthikeyan Kandavelou, Jeremy M. Berg, Srinivasan Chandrasegaran,