Nickel-mediated sequence-specific oxidative cleavage of DNA by a designed metalloprotein

We recently designed a sequence-specific DNA-cleaving metalloprotein consisting wholly of naturally occurring α-amino acids. The tripeptide copper-binding domain, Gly-Gly-His (GGH), was attached to the NH2-terminus of the DNA-binding domain of Hin recombinase (residues 139-1 90) to afford a new 55-residue protein, GGH(Hin 139-1 90). This protein is capable of binding DNA at four 13 base pair sites (termed hixL and secondary). CuDGGH(Hin139-190) in the presence of excess hydrogen peroxide and sodium ascorbate cleaves DNA predominantly at one of the four Hin binding sites. We report here that, in the presence of Ni(OAc), and monoperoxyphthalic acid, the sequence specificity and efficiency of the DNA cleavage by GGH(Hin139-190) are remarkably altered. The nickel-mediated DNA cleavage process occurs at all four binding sites, is more rapid and efficient, and is chemically activated by 1 equiv of an oxygen atom donor. At the hixL site, cleavage occurs predominantly at a single deoxyribose position on one strand of each binding site, indicating a nondiffusible oxidizing species.