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Use of Oligodeoxynucleotides Containing O⁶-Alkylguanine for the Assay of O⁶-Alkylguanine-DNA-alkyltransferase Activity¹

Department of Physiology and Cancer Research Center, Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

A sensitive assay procedure was developed for the measurement of the activity of mammalian O⁶-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O⁶-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with ³²P by the reaction with polynucleotide kinase and [-³²P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase 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 alkyltransferase 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 alkyltransferase 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 alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

¹ Supported by NIH Grant CA-18137.

² To whom requests for reprints should be addressed.

Received 7/ 8/87. Revised 10/30/87. Accepted 12/ 3/87.