A new assay for O6-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 greater than 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 inter-individual 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.
Klein
S
S
Institute of Toxicology, University of Mainz, FRG.
Oesch
F
F
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004260:DNA Repair; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology*; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013379:Substrate Specificity
D004279:DNA, Viral; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase