Excision of O6-methylguanine from DNA by human fibroblasts determined by a sensitive competition method.
A new, simple and relatively inexpensive assay for measuring O6-methylguanine (O6-MeG) in methylated DNA is described. This assay used 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 molecular has been shown to act only once. In our assay, 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 favourably with current radiochemical and immunological techniques. We have used this assay to measure O6-MeG produced in the DNA of human fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. We have also studied the kinetics of removal of this lesion. We confirm previous reports of a rapid repair system for the removal of O6-MeG from DNA by human fibroblasts.
D004247:DNA / Q000032:analysis*; D005347:Fibroblasts / Q000032:analysis; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D008769:Methylnitronitrosoguanidine; D008780:Methyltransferases / Q000037:antagonists & inhibitors
D008769:Methylnitronitrosoguanidine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D008780:Methyltransferases