Assay by inhibition of repair to measure O6-methylguanine in DNA.
A procedure for measuring the level of O6-methylguanine (O6-meG) in DNA is described. Repair of 32P-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O6-meG. Each methylated DNA sample inhibited the repair of oligodeoxynucleotide substrate to an extent dependent upon O6-meG concentration. Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O6-meG concentration. We report the method of calculation of the O6-meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs. This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used. The method requires only 0.1-10 micrograms of DNA, with a limit of detection of 0.8 fmol of O6-meG per microgram of DNA.
Mironov
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NM
Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Martel-Planche
G
G
Wild
C P
CP
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D004926:Escherichia coli; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011863:Radioimmunoassay
D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase