Assay for O6-alkylguanine-DNA-alkyltransferase using oligonucleotides containing O6-methylguanine in a BamHI recognition site as substrate.
Double-stranded oligonucleotides, 40 bases in length containing an O6-methylguanine in a BamHI restriction site, were developed as substrates for the determination of human O6-alkylguanine-DNA-alkyltransferase (AGT). The assay proved highly sensitive and quantitative. After incubation of the 5'-end-labeled oligonucleotides with cell homogenates of peripheral blood lymphocytes, the DNA was digested with BamHI. Cleavage with this restriction enzyme did not occur in the O6-methylguanine-containing oligonucleotide unless the fragment was repaired. The cleaved oligonucleotide was separated from the intact parent oligonucleotide by reverse-phase high-performance liquid chromatography. Calculation of the AGT content was achieved by integrating the radioactivity of the peak corresponding to the digested fragment, which is equal to the molar amount of repaired oligonucleotide and corresponds directly to the molar AGT content in the lymphocyte homogenate.
Klein
S
S
Institute of Toxicology, University of Mainz, Germany.
Oesch
F
F
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D008780:Methyltransferases / Q000032:analysis*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009841:Oligonucleotides / Q000032:analysis* / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000032:analysis; D010761:Phosphorus Radioisotopes; D012680:Sensitivity and Specificity
D009841:Oligonucleotides; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase