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

The protein O-6-alkylguanine-DNA alkyltransferase (alkyltransferase) is inv olved in the repair of O-6-alkylguanine and O-4-alkylthymine in DNA and pla ys an important role in most organisms in attenuating the cytotoxic and mut agenic effects of certain classes of alkylating agents. A genomic clone enc ompassing the Drosophila melanogaster alkyltransferase gene (DmAGT) was ide ntified on the basis of sequence homology with corresponding genes in Sacch aromyces cerevisiae and man. The DmAGT gene is located at position 84A on t he third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmA GT cDNA. Furthermore, alkyltransferase activity was identified in crude ext racts of Escherichia coil harbouring DmAGT cDNA and this activity was inhib ited by preincubation of the extract with an oligonucleotide containing a s ingle O-6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransf erase but in contrast to the human alkyltransferase, the Drosophila alkyltr ansferase is resistant to inactivation by O-6-benzylguanine. In an E.coli l acZ reversion assay, expression of DmAGT efficiently suppressed MNNG-induce d G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These result s demonstrate the presence of an alkyltransferase specific for the repair o f O-6-methylguanine and O-4-methylthymine in Drosophila.