Cloning of a 3-methyladenine-DNA glycosylase from Arabidopsis thaliana.

We have isolated an Arabidopsis thaliana cDNA that complements the methyl methanesulfonate-sensitive phenotype of an Escherichia coli double mutant deficient in 3-methyladenine glycosylases (DNA-3-methyladenine glycosidases I and II, EC 3.2.2.20 and 3.2.2.21, respectively, encoded by tag and alkA). Expression of the Arabidopsis cDNA enhances the methyl methanesulfonate resistance of the E. coli double mutant by nearly four orders of magnitude. The cDNA corresponds to a single-copy, nuclearly encoded sequence which specifies a predicted 28.1-kDa protein with a charge of +8 at pH 7.0. Enzymatic analysis of extracts prepared from the transformed mutants indicates that the cDNA encodes a 3-methyladenine glycosylase. The predicted amino acid sequence of the Arabidopsis glycosylase has significant homology to other eukaryotic 3-methyladenine glycosylases. IMAGES: