Use of oligodeoxynucleotides containing o⁶-alkylguanine for the assay of o⁶-alkylguanine-dna-alkyl transferase activity

A sensitive assay procedure was developed for the measurement of the activity of mammalian O⁶-alkylguanine-DNA-alkyl transferase. The procedure utilized oligodeoxynucleotides containing O⁶-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with ³² P by the reaction with polynucleotide kinase and [γ-³² P]ATP and allowed to react with organ or cell extracts containing the alkyl transferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyl transferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyl transferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyl transferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyl transferase activity in samples in which the activity is very low or the amount of material available is limited.