ASSAY BY INHIBITION OF REPAIR TO MEASURE O6-METHYLGUANINE IN DNA

A procedure for measuring the level of O6-methylguanine (O6-meG) in DN A is described. Repair of P-32-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in th e presence of different quantities of DNA containing unknown concentra tions of O6-meG. Each methylated DNA sample inhibited the repair of ol igodeoxynucleotide substrate to an extent dependent upon O6-meG concen tration. Each DNA sample tested at different concentrations in the ass ay therefore had a characteristic inhibition curve and could be compar ed to the curves generated using reference DNA samples of known O6-meG concentration. We report the method of calculation of the O6-meG leve l in a given DNA sample by comparison of its inhibition curve with tha t of reference DNAs. This method of calculation does not require a kno wledge of the exact quantity of the labelled substrate or AGT used. Th e method requires only 0.1-10 mug of DNA, with a limit of detection of 0.8 fmol of O6-meG per mug of DNA. (C) 1994 Wiley-Liss, Inc.