Stopped-flow kinetic studies of the interaction between Escherichia coli Fpg protein and DNA substrates.
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 nucleotide in 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.
Fedorova
Olga S
OS
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk. fedorova@niboch.nsc.ru
Nevinsky
Georgy A
GA
Koval
Vladimir V
VV
Ishchenko
Alexander A
AA
Vasilenko
Nataly L
NL
Douglas
Kenneth T
KT
D001665:Binding Sites; D002384:Catalysis; D004269:DNA, Bacterial / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors
D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; C024829:8-hydroxyguanine; D006147:Guanine; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli