title: Real-time fluorescence assay for O6-alkylguanine-DNA alkyltransferase.
abstract: O6-alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6-position of guanine. We developed a real-time AGT assay that utilizes a fluorescent guanosine analog (3-methylisoxantopterin, 3-MI). 3-MI fluorescence is quenched in DNA and fluorescence intensity increases substantially with digestion of the oligonucleotide and release of 3-MI. The substrate is a doubled-stranded oligonucleotide with 3'-overhangs on each end and a PvuII recognition site. PvuII is inhibited by O6-methylguanine, positioned within the restriction site. 3-MI is incorporated in the opposite strand just outside of the PvuII restriction site. AGT repairs O6-methylguanine; PvuII cleaves at its restriction site, yielding a blunt-ended double strand, which is then digested by exonuclease III. This releases 3-MI from the oligonucleotide, resulting in an increase in fluorescence intensity. All reaction components (100-microL volume) are monitored in a single microcuvette. Rate of increase in fluorescence intensity is related to the amount of AGT in the reaction mixture. We measured AGT levels in extracts from a leukemia cell line, from leukemic lymphoblasts from patients, and from peripheral blood mononuclear cells from normal controls. This method may prove useful for mechanistic studies of AGT.
authors.affiliation: Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Moser
authors.forename: M
authors.initials: M
authors.lastname: Patel
authors.forename: H
authors.initials: H
authors.lastname: Yoo
authors.forename: F M
authors.initials: FM
authors.lastname: Balis
authors.forename: M E
authors.initials: ME
authors.lastname: Hawkins
mesh_terms: D001483:Base Sequence; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis*; D009841:Oligonucleotides; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D009841:Oligonucleotides; D011994:Recombinant Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase