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

M. Eileez Dolan, David Scicchitano, Anthony Pegg

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1184-1188
Number of pages5
JournalCancer Research
Volume48
Issue number5
StatePublished - Jan 1 1988

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Oligodeoxyribonucleotides
Transferases
Polynucleotide 5'-Hydroxyl-Kinase
Reverse-Phase Chromatography
Cell Extracts
Adenosine Triphosphate
High Pressure Liquid Chromatography
Cell Line
DNA
Proteins

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

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title = "Use of oligodeoxynucleotides containing o6-alkylguanine for the assay of o6-alkylguanine-dna-alkyl transferase activity",
abstract = "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.",
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Use of oligodeoxynucleotides containing o6-alkylguanine for the assay of o6-alkylguanine-dna-alkyl transferase activity. / Eileez Dolan, M.; Scicchitano, David; Pegg, Anthony.

In: Cancer Research, Vol. 48, No. 5, 01.01.1988, p. 1184-1188.

Research output: Contribution to journalArticle

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AB - 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.

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