Human endonuclease III acts preferentially on DNA damage opposite guanine residues in DNA

The human endonuclease III homologue (hNTH1) removes premutagenic cytosine damage from DNA. This includes 5-hydroxycytosine, which has increased poten tial for pairing with adenine, resulting in C --> T transition mutations. H ere we report that hNTH1 acts on both 5-hydroxycytosine and abasic sites pr eferentially when these are situated opposite guanines in DNA. Discriminati on against other opposite bases is strongly dependent on the presence of ma gnesium. To further elucidate this effect, we have introduced mutations in the helix-hairpin-helix domain of hNTH1 (K212S, P211R, +G212, and Delta P21 1), and measured the kinetics of 5-hydroxycytosine removal of the mutants r elative to wild type. The K212S and Delta P211 (truncated hairpin) mutant p roteins were both inactive, whereas the extended hairpin in the +G212 mutan t diminished recognition and binding to 5-hydroxycytosine-containing DNA. T he P211R mutant resembled native hNTH1, except for decreased specificity of binding. Despite the altered kinetic parameters, the active mutants retain ed the ability to discriminate against the pairing base, indicating that en zyme interactions with the opposite strand relies on other domains than the active site helix-hairpin-helix motif.