title: Redesign of high-affinity nonspecific nucleases with altered sequence preference.
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 protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Wang
authors.forename: Jon D
authors.initials: JD
authors.lastname: Wright
authors.forename: Lyudmila G
authors.initials: LG
authors.lastname: Doudeva
authors.forename: Hua-Ci
authors.initials: HC
authors.lastname: Jhang
authors.forename: Carmay
authors.initials: C
authors.lastname: Lim
authors.forename: Hanna S
authors.initials: HS
authors.lastname: Yuan
mesh_terms: D003086:Bacteriocin Plasmids / Q000737:chemistry / Q000378:metabolism*; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular
chemical_list: D004798:Enzymes