Identification and characterisation of theDrosophila melanogaster O 6-alkylguanine-DNA alkyltransferase cDNA

The protein O6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O6-alkylguanine and O4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene (DmAGT) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficientbacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O6-benzylguanine. In an E.coli lacZ reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C[Right arrow]A:T as well as A:T[Right arrow]G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O6-methylguanine and O4-methylthymine in Drosophila.