A novel fluorometric oligonucleotide assay to measure O-6-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

DNA repair status plays a major role in mutagenesis, carcinogenesis and res istance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required seve ral assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O-6-methylguanine DNA met hyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeas t and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a s ingle 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 l esions specific to each repair protein, This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sa mple. In addition, the stability of the fluorometric oligonucleotides precl udes the substrate variability caused by continual radiolabeling, In this r eport this technique was applied to human breast carcinoma MDA-MB231 cells over-expressing human MPG in order to assess whether up-regulation of the i nitial step in BER alters the activity of selected other BER (hOGG1 and APE /ref-1) or direct reversal (MGMT) repair activities.