Stopped-flow kinetic studies of the interaction between Escherichia coli Fpg protein and DNA substrates

被引:58
作者
Fedorova, OS
Nevinsky, GA
Koval, VV
Ischenko, AA
Vasilenko, NL
Douglas, KT [1 ]
机构
[1] Russian Acad Sci, Siberian Div, Novosibirsk Bioorgan Chem Inst, Novosibirsk 630090, Russia
[2] Univ Manchester, Sch Pharm & Pharmaceut Sci, Manchester M13 9PL, Lancs, England
关键词
D O I
10.1021/bi011524u
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Formamidopyrimidine-DNA-glycosylase of Escherichia coli (Fpg protein) repairs oxidative DNA damage by removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. This enzyme possesses three types of activities resulting in the excision of oxidized residue from DNA: hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP-lyase), and delta-elimination. In our work, the kinetic mechanism for 8-oxoguanine excision from DNA substrate with Fpg protein has been determined from stopped-flow measurements of changes in the tryptophan fluorescence. The 12-nucleotide duplex d(CTCTC(oxo)GCCTTCC).d(GGAAGGCGAGAG) containing the 8-oxoG nuclcotide ill the sixth position of one strand was used as the specific substrate. Four distinct phases in the time traces were detected. These four-phase transition changes in the Fpg protein fluorescence curves were analyzed by global fitting to determine the intrinsic rate constants. We propose that the first two phases represent the equilibrium steps. The first of them describes the bimolecular binding step and the second, formation of the apurinic site. The third, irreversible step is believed to describe the beta-elimination process. The fourth step reflects the delta-elimination and decomposition of complex between enzyme and the product of 8-oxoG nucleotide excision. The results obtained provide direct evidence of conformational transitions of the Fpg protein during the catalytic process. The significance of these results for the functioning of Fpg protein is discussed.
引用
收藏
页码:1520 / 1528
页数:9
相关论文
共 34 条
[21]   The reaction mechanism of DNA glycosylase/AP lyases at abasic sites [J].
McCullough, AK ;
Sanchez, A ;
Dodson, ML ;
Marapaka, P ;
Taylor, JS ;
Lloyd, RS .
BIOCHEMISTRY, 2001, 40 (02) :561-568
[22]   Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily [J].
Nash, HM ;
Bruner, SD ;
Scharer, OD ;
Kawate, T ;
Addona, TA ;
Sponner, E ;
Lane, WS ;
Verdine, GL .
CURRENT BIOLOGY, 1996, 6 (08) :968-980
[23]   DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required [J].
Picha, KM ;
Ahnert, P ;
Patel, SS .
BIOCHEMISTRY, 2000, 39 (21) :6401-6409
[24]   Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae [J].
Radicella, JP ;
Dherin, C ;
Desmaze, C ;
Fox, MS ;
Boiteux, S .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (15) :8010-8015
[25]   NORMAL OXIDATIVE DAMAGE TO MITOCHONDRIAL AND NUCLEAR-DNA IS EXTENSIVE [J].
RICHTER, C ;
PARK, JW ;
AMES, BN .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (17) :6465-6467
[26]   Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase [J].
RoldanArjona, T ;
Wei, YF ;
Carter, KC ;
Klungland, A ;
Anselmino, C ;
Wang, RP ;
Augustus, M ;
Lindahl, T .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (15) :8016-8020
[27]   8-hydroxyguanine (7,8-dihydro-8-oxoguanine) DNA glycosylase and AP lyase activities of hOGG1 protein and their substrate specificity [J].
Shinmura, K ;
Kasai, H ;
Sasaki, A ;
Sugimura, H ;
Yokota, J .
MUTATION RESEARCH-DNA REPAIR, 1997, 385 (01) :75-82
[28]   MEASUREMENT OF PROTEIN USING BICINCHONINIC ACID [J].
SMITH, PK ;
KROHN, RI ;
HERMANSON, GT ;
MALLIA, AK ;
GARTNER, FH ;
PROVENZANO, MD ;
FUJIMOTO, EK ;
GOEKE, NM ;
OLSON, BJ ;
KLENK, DC .
ANALYTICAL BIOCHEMISTRY, 1985, 150 (01) :76-85
[29]   STUDIES ON THE CATALYTIC MECHANISM OF 5 DNA GLYCOSYLASES - PROBING FOR ENZYME-DNA IMINO INTERMEDIATES [J].
SUN, B ;
LATHAM, KA ;
DODSON, ML ;
LLOYD, RS .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (33) :19501-19508
[30]   THE CATALYTIC MECHANISM OF FPG PROTEIN - EVIDENCE FOR A SCHIFF-BASE INTERMEDIATE AND AMINO-TERMINUS LOCALIZATION OF THE CATALYTIC SITE [J].
TCHOU, J ;
GROLLMAN, AP .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (19) :11671-11677