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

被引:25
作者
Moser, AM
Patel, M
Yoo, H
Balis, FM
Hawkins, ME [1 ]
机构
[1] Natl Canc Inst, Pediat Oncol Branch, Bethesda, MD 20892 USA
[2] Childrens Canc Ctr, San Antonio, TX 78229 USA
关键词
real-time DNA-repair assay; fluorescent nucleoside analog; alkylation; pteridine;
D O I
10.1006/abio.2000.4554
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
O-6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that reverses 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 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 PuvII recognition site. PvuII is inhibited by Op-methylguanine, positioned within the restriction site. 3-MI is incorporated in the opposite strand just outside of the PvuII restriction site. AGT repairs O-6-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-mu L 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.
引用
收藏
页码:216 / 222
页数:7
相关论文
共 19 条
[1]  
Belanich M, 1996, CANCER RES, V56, P783
[2]  
DOLAN ME, 1988, CANCER RES, V48, P1184
[3]   DEMETHYLATION OF O-6-METHYLGUANINE IN A SYNTHETIC DNA POLYMER BY AN INDUCIBLE ACTIVITY IN ESCHERICHIA-COLI [J].
FOOTE, RS ;
MITRA, S ;
PAL, BC .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1980, 97 (02) :654-659
[4]  
Futscher B W, 1989, Cancer Commun, V1, P65
[5]  
GERSON SL, 1989, CANCER RES, V49, P3134
[6]   INCORPORATION OF A FLUORESCENT GUANOSINE ANALOG INTO OLIGONUCLEOTIDES AND ITS APPLICATION TO A REAL-TIME ASSAY FOR THE HIV-1 INTEGRASE 3'-PROCESSING REACTION [J].
HAWKINS, ME ;
PFLEIDERER, W ;
MAZUMDER, A ;
POMMIER, YG ;
FALLS, FM .
NUCLEIC ACIDS RESEARCH, 1995, 23 (15) :2872-2880
[7]   Fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into oligonucleotides [J].
Hawkins, ME ;
Pfleiderer, W ;
Balis, FM ;
Porter, D ;
Knutson, JR .
ANALYTICAL BIOCHEMISTRY, 1997, 244 (01) :86-95
[8]   Correlation of tumor O6 methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea:: A Southwest Oncology Group study [J].
Jaeckle, KA ;
Eyre, HJ ;
Townsend, JJ ;
Schulman, S ;
Knudson, HM ;
Belanich, M ;
Yarosh, DB ;
Bearman, SI ;
Giroux, DJ ;
Schold, SC .
JOURNAL OF CLINICAL ONCOLOGY, 1998, 16 (10) :3310-3315
[9]   ASSAY FOR O6-ALKYLGUANINE-DNA-ALKYLTRANSFERASE USING OLIGONUCLEOTIDES CONTAINING O6-METHYLGUANINE IN A BAMHI RECOGNITION SITE AS SUBSTRATE [J].
KLEIN, S ;
OESCH, F .
ANALYTICAL BIOCHEMISTRY, 1992, 205 (02) :294-299
[10]  
OLSSON M, 1980, J BIOL CHEM, V260, P2605