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, Delta QNK-FN, in order to gain insight into how ZFNs cleave DNA and how two. inverted sites promote double-strand cleavage. DNA cleavage by Delta QNK-FN is greatly facilitated when two Delta QNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of Delta QNK-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 Delta QNK-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 Delta QNK-FN active complex required both binding of the two Delta QNK-FN molecules to specific DNA sites and divalent metal ions. (c) 2005 Elsevier Inc. All rights reserved.