Real-time fluorescence assay for O-6-alkylguanine-DNA alkyltransferase

O-6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that re verses the effects of alkylating agents by removing DNA adducts from the O- 6-position of guanine, We developed a real-time AGT assay that utilizes a f luorescent guanosine analog (3-methylisoxantopterin, 3-MI). 3-MI fluorescen ce is quenched in DNA and fluorescence intensity increases substantially wi th digestion of the oligonucleotide and release of 3-MI. The substrate is a doubled-stranded oligonucleotide with 3'-overhangs on each end and a PuvII recognition site. PvuII is inhibited by Op-methylguanine, positioned withi n the restriction site. 3-MI is incorporated in the opposite strand just ou tside of the PvuII restriction site. AGT repairs O-6-methylguanine; PvuII c leaves at its restriction site, yielding a blunt-ended double strand, which is then digested by exonuclease III. This releases 3-MI from the oligonucl eotide, resulting in an increase in fluorescence intensity. All reaction co mponents (100-mu L volume) are monitored in a single microcuvette. Rate of increase in fluorescence intensity is related to the amount of AGT in the r eaction mixture. We measured AGT levels in extracts from a leukemia cell li ne, from leukemic lymphoblasts from patients, and from peripheral blood mon onuclear cells from normal controls, This method may prove useful for mecha nistic studies of AGT, (C) 2000 Academic Press.