A 55-residue protein containing the DNA binding domain of Hin recombinase, residues 139-190, with the tripeptide Gly-Gly-His (GGH) at the NH2 terminus was synthesized by stepwise solid-phase methods. GGH(Hin139-190) binds sequence specifically to DNA at four 13 base pair sites (termed hixL and secondary) and, in the presence of Ni(OAc)2 and monoperoxyphthalic acid, reacts predominantly at a single deoxyribose position on one strand of each binding site [Mack, D.P., & Dervan, P.B. (1990) J. Am. Chem. Soc. 112, 4604]. We find that, upon treatment with n-butylamine, the DNA termini at the cleavage site are 3'- and 5'-phosphate, consistent with oxidative degradation of the deoxyribose backbone. The nickel-mediated oxidation can be activated with peracid, iodosylbenzene, or hydrogen peroxide. The sequence specificity of the reaction is not dependent on oxidant, but the rates of cleavage differ, decreasing in the order peracid greater than iodosylbenzene greater than hydrogen peroxide. Optimal cleavage conditions for a 1 microM concentration of protein are 50 microM peracid, pH 8.0, and 1 equiv of Ni(OAc)2. The preferential cleavage at a single base pair position on one strand of the minor groove indicates a nondiffusible oxidizing species. A change of absolute configuration in the GGH metal binding domain from L-His to D-His [Ni(II).GG-(-D-)H(Hin139-190)] affords cleavage at similar base pair locations but opposite with regard to strand specificity.