Assay by inhibition of repair to measure O6-methylguanine in DNA

A procedure for measuring the level of O⁶-me-thylguanine (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 oli-godeoxynucleotide 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 μg of DNA, with a limit of detection of 0.8 fmol of O⁶-meG per μg of DNA. © 1994 Wiley-Liss, Inc.