Sequence-selective and hydrolytic cleavage of DNA by zinc finger mutants |
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Authors: | Nomura Akiko Sugiura Yukio |
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Institution: | Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. |
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Abstract: | We have reported the successful conversion of the structural zinc site in zinc finger peptides to a functional zinc site. A series of resulting zinc finger mutants exhibit the hydrolytic ability of the activated ester depending on the coordination geometry and acidity of the zinc ions. In this study, we explored the hydrolytic ability of DNA by the H4 mutant since the mutant showed the highest hydrolytic ability of the activated ester among the series of mutant peptides. The zinc-bound form of the H4 mutant peptide exhibited the hydrolytic ability of activated phosphoesters and even converted the supercoiled plasmid to the nicked circular form. An increasing ionic strength leads to a loss in the nuclease ability of the zinc finger mutants due to the nonspecific interaction between the zinc finger peptide and DNA. In sharp contrast, the three-tandem H4-type zinc finger protein performed the specific DNA hydrolysis at the GC box even at a high ionic strength. Thus, the present study demonstrated that converting the native zinc site to the hydrolytic zinc site in the zinc finger protein is a novel approach for creating artificial nucleases with sequence selectivity. |
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