Structural basis for enzymatic excision of N 1 ‐methyladenine and N 3 ‐methylcytosine from DNA

N 1 ‐methyladenine (m 1 A) and N 3 ‐methylcytosine (m 3 C) are major toxic and mutagenic lesions induced by alkylation in single‐stranded DNA. In bacteria and mammals, m 1 A and m 3 C were recently shown to be repaired by AlkB‐mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m 1 A and m 3 C are repaired by the Af AlkA base excision repair glycosylase of Archaeoglobus fulgidus , suggesting a different repair mechanism for these lesions in the third domain of life. In addition, Af AlkA was found to effect a robust excision of 1, N 6 ‐ethenoadenine. We present a high‐resolution crystal structure of Af AlkA, which, together with the characterization of several site‐directed mutants, forms a molecular rationalization for the newly discovered base excision activity.