A NEW ASSAY FOR O-6-ALKYLGUANINE-DNA-ALKYLTRANSFERASE TO DETERMINE DNA-REPAIR CAPACITIES USING LAMBDA-PHAGE DNA AS SUBSTRATE

One O6-methylguanine (O6-meG) was introduced into each BamHI site of lambda-phage DNA as a substrate for the determination of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase. A new assay using as the detection group 32P-labeled phosphate introduced at the 3' position of the modified nucleoside by incorporation of 32P-labeled TTP in the 3'-neighboring position proved highly sensitive: 10-16 mol of the DNA lesion was still easily detectable. This DNA, which has >1000 bp represents a good model for cellular DNA and was used as a substrate to measure the individual repair capacities for O6-meG in human lymphocytes of 20 healthy male and female donors. There were great interindividual variations in the activity of this repair protein in man. The influences of age, sex or smoking behavior on the repair capacity of O6-meG were negligible. © 1990 Oxford University Press.