Structure–function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix–hairpin–helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N⁶-ethenoadenine ([epsilon]A) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-­methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-­glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.