O (6)-Alkylguanine-DNA Alkyltransferase Assay

Alkylating agents exert a wide range of biological effects in both pro- and eukaryotes and there is ever increasing evidence that these effects are mediated via alkylation at the O (6)position of guanine in DNA (1-4). Repair of such adducts can be mediated by O (6)-alkylguanine-DNA alkyltransferase (ATase) (3,4). Both pro- and eukaryote ATases transfer alkyl groups from the O (6)-position of guanine in alkylated DNA (or from other low molecular weight substrates); (5) to a cysteine residue located at the active site of the protein: the reaction is stoichiometric and the protein is autoinactivated (6). This mechanism has been exploited in the design of several different radioactivity-based assays for the enzyme. These involve either measurement of methyl group transfer to protein or the analysis (e.g., by HPLC) of methylated substrate DNA before and after exposure to cell or tissue extracts or restriction endonuclease (RE) site deprotection of synthetic oligonucleotide substrates containing O (6)-methylguanine.