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 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 O(6)-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic 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 overexpressing 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.
Kreklau
E L
EL
Department of Pharmacology, Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Limp-Foster
M
M
Liu
N
N
Xu
Y
Y
Kelley
M R
MR
Erickson
L C
LC
D000478:Alkylation; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics; D002021:Buffers; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D045647:DNA Glycosylases; D019175:DNA Methylation; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005453:Fluorescence; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020014:K562 Cells; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002021:Buffers; D009841:Oligonucleotides; D019853:O(6)-Methylguanine-DNA Methyltransferase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase