TY - JOUR
T1 - Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage
AU - Mani, Mala
AU - Smith, Jeff
AU - Kandavelou, Karthikeyan
AU - Berg, Jeremy M.
AU - Chandrasegaran, Srinivasan
N1 - Funding Information:
We thank Dr. Neil Clarke for helpful suggestions and comments on the kinetics experiments. This work was funded by a grant from National Institutes of Health (GM 53923). We also thank Environmental Health Sciences Center Core Facility (supported by Grant ES 03819) for the synthesis of oligonucleotides. We thank Dr. Sundar Durai for help with figures. K.K. was supported for a year by the Johns Hopkins University Center for AIDS Research (CFAR; Grant # P30 AI42855) grant.
PY - 2005/9/9
Y1 - 2005/9/9
N2 - 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.
AB - 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.
KW - Chimeric nucleases
KW - Directed mutagenesis
KW - Gene targeting
KW - Gene therapy
KW - Homologous recombination
KW - Homology-directed repair
KW - Non-homologous end joining
KW - Protein engineering
KW - Sequence-specific cleavage
KW - Zinc finger nucleases
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U2 - 10.1016/j.bbrc.2005.07.021
DO - 10.1016/j.bbrc.2005.07.021
M3 - Article
C2 - 16043120
AN - SCOPUS:23044460704
SN - 0006-291X
VL - 334
SP - 1191
EP - 1197
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -