Redesign of high-affinity nonspecific nucleases with altered sequence preference

Journal of the American Chemical Society
Yi-Ting WangHanna S Yuan

Abstract

It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the pr...Continue Reading

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Citations

Sep 3, 2014·Metallomics : Integrated Biometal Science·Anikó CzeneBéla Gyurcsik
Oct 26, 2011·Future Medicinal Chemistry·Béla Gyurcsik, Anikó Czene
Aug 27, 2014·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Eszter NémethBéla Gyurcsik
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May 23, 2013·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Anikó CzeneKyosuke Nagata

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