Cloning of the E. coli O6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay

Abstract

Alkylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates for repair processes. Oxygen atom derivatives such as O6-methylguanine (O6-meG) O4-methylthymine and methylphosphotriesters (MP) have been shown to undergo repair by methyl group removal. The proteins involved in the latter reaction can be considered to be methyltransferases (MT) because their action results in the transfer of the methyl group to a cysteine residue within a polypeptide. A rapid and sensitive assay for MT activity has been developed and used to screen extracts of bacteria harbouring an E. coli genomic DNA library carried in a plasmid vector. We report here the cloning of an E. coli gene coding for O6-meG and MP MT repair functions. These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an 18Kd protein which contains only O6-meG MT and a 13Kd protein which contains only MP MT.