Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage
| Autor: | Jeremy M Berg, Srinivasan Chandrasegaran, Mala Mani, Jeffrey S. Smith, Karthikeyan Kandavelou |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Cleavage factor
DNA damage Biophysics Cleavage and polyadenylation specificity factor Biology Cleavage (embryo) Biochemistry Article chemistry.chemical_compound Molecular Biology Zinc finger Binding Sites Deoxyribonucleases Zinc Fingers DNA Cell Biology Zinc finger nuclease DNA-Binding Proteins Enzyme Activation DNA binding site chemistry DNA Damage Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 334:1191-1197 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2005.07.021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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