A novel fluorometric oligonucleotide assay to measure O6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase

Emiko L. Kreklau, Melissa Limp-Foster, Naili Liu, Yi Xu, Mark Kelley, Leonard C. Erickson

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O6-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human a basic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells over-expressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.

Original languageEnglish
Pages (from-to)2558-2566
Number of pages9
JournalNucleic Acids Research
Volume29
Issue number12
StatePublished - Jun 15 2001

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DNA Glycosylases
Endonucleases
Methyltransferases
Oligonucleotides
DNA Repair
Breast Neoplasms
DNA
Cell Extracts
8-hydroxyguanine
O-(6)-methylguanine
Alkylation
Mutagenesis
Methylation
Buffers
Carcinogenesis
Proteins
Up-Regulation
Yeasts

ASJC Scopus subject areas

  • Genetics

Cite this

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abstract = "DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O6-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human a basic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells over-expressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.",
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AU - Kreklau, Emiko L.

AU - Limp-Foster, Melissa

AU - Liu, Naili

AU - Xu, Yi

AU - Kelley, Mark

AU - Erickson, Leonard C.

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