Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage.
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, DeltaQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by DeltaQNK-FN is greatly facilitated when two DeltaQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of DeltaQNK-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 DeltaQNK-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 DeltaQNK-FN active complex required both binding of the two DeltaQNK-FN molecules to specific DNA sites and divalent metal ions.
Mani
Mala
M
Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
Smith
Jeff
J
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
Kandavelou
Karthikeyan
K
Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
Berg
Jeremy M
JM
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
Chandrasegaran
Srinivasan
S
Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003851:Deoxyribonucleases / Q000737:chemistry*; D004789:Enzyme Activation; D011485:Protein Binding; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D004247:DNA; D003851:Deoxyribonucleases