Cloning of theE. coliO6-methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay

Abstract

Akylating agents react with various nitrogen and oxygen atoms in DNA and many of the products are substrates repair processes. Oxygen atom derivatives such as 0 ⁶ -methylguanine ( 0 ⁶ -meG) 0 ⁴ -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 methyltranferases (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 glasmid vector. We report here the cloning of an E.coli gene coding for 0 ⁶ -meG and MP MT repair functions. These two activities reside on a 37Kd protein that can undergo a host-dependent cleavage to produce an l8Kd protein which contains only 0 ⁶ -meG MT and a l3Kd protein which contains only MP MT.