Excision of O6-methylguanine from DNA by human fibroblasts determined by a sensitive competition method

Abstract

A new, simple and relatively inexpensive assay for measuring O6-methylguanine (O6-MeG) in methylated DNA is described. This assay uses the property of suicide inactivation of Escherichia coli methyltransferase. When crude extracts of methyltransferase are incubated with DNA containing O6-MeG, transfer of the methyl group to a cysteine residue on the protein occurs and the protein is subsequently inactivated. Each protein molecule was shown to act only once. Methylated DNA is first incubated with a fixed amount of the extract. The O6-MeG present in this DNA proportionally inactivates a part of the methyltransferase activity. The remaining activity is then incubated with a fixed amount of O6-[3H]MeG substrate of known specific activity and precipitated with acid. Hydrolysis of the acid precipitable fraction releases the unaltered O6-[3H]MeG enabling the amount of O6-MeG present in the unknown sample to be calculated. This method enables the accurate measurement of as little as 0.1 pmol of O6-MeG in methylated DNA and compares favorably with current radiochemical and immunological techniques. This assay was used to measure O6-MeG produced in the DNA of human fibroblasts treated with N-methyl-N''-nitro-N-nitrosoguanidine. The kinetics of removal of this lesion were studied. Previous reports of a rapid repair system for the removal of O6-MeG from DNA by human fibroblasts were confirmed. [This study may be applicable to carcinogenesis.].