Characterisation and nucleotide sequence ofogt, the O6-alkyiguanine-DNA-alkyltransferase gene ofE.coli

Abstract

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.