Characterisation and nucleotide sequence of ogt, the O-alkyiguanine-DNA-alkyltransferase gene of E.coli

The plasmid p061 that was isolated fran an E.coli genomic DNA library and codes for 06-alkylguanine (06 AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of p061 showed that the ATase activity was encoded in a 2Kb Pstl but a partial restriction endonuclease map of this was different to that of the E.coli ada gene that codes for 06 -AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the p061-encoded ATase was 19KDa i.e. similar to that of the 06 AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the p061-encoded protein. A different set of hybridisation signals was produced when E.coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E.coli. The 2Kb Pst-1 fragment of p061 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the 06 AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada 06 AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and SI nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.