Real-time fluorescence assay for O⁶-alkylguanine-DNA alkyltransferase

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-μL 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. (C) 2000 Academic Press.