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. 2004 Feb 9;32(3):926-35.
doi: 10.1093/nar/gkh237. Print 2004.

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Vladimir V Koval  1 , Nikita A Kuznetsov, Dmitry O Zharkov, Alexander A Ishchenko, Kenneth T Douglas, Georgy A Nevinsky, Olga S Fedorova
Affiliations

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Vladimir V Koval et al. Nucleic Acids Res. .

Abstract

Formamidopyrimidine-DNA-glycosylase (Fpg protein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2'-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.

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Figures

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Scheme 1. Fpg binding to an F-containing duplex. F, F-containing ligand; (E·F)i, different enzyme–ligand complexes.
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Scheme 1. Fpg binding to an F-containing duplex. F, F-containing ligand; (E·F)i, different enzyme–ligand complexes.
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Scheme 2. Binding and cleavage of an AP-containing substrate by Fpg. AP, AP-containing substrate; (E·AP)i, different enzyme–substrate complexes; Pgap, an immediate product of substrate conversion, i.e. a gapped DNA duplex; Pmix, Pgap dissociated into three individual oligonucleotides; Kp is the combined dissociation constant for the release of Pgap and its dissociation to Pmix.
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Scheme 2. Binding and cleavage of an AP-containing substrate by Fpg. AP, AP-containing substrate; (E·AP)i, different enzyme–substrate complexes; Pgap, an immediate product of substrate conversion, i.e. a gapped DNA duplex; Pmix, Pgap dissociated into three individual oligonucleotides; Kp is the combined dissociation constant for the release of Pgap and its dissociation to Pmix.
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Scheme 3. Binding and cleavage of an 8-oxoG-containing substrate by Fpg. OG, 8-oxoG-containing substrate, (E·OG)i, different enzyme–substrate complexes.
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Scheme 3. Binding and cleavage of an 8-oxoG-containing substrate by Fpg. OG, 8-oxoG-containing substrate, (E·OG)i, different enzyme–substrate complexes.
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Scheme 4. Binding and cleavage of any substrate by Fpg.
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Scheme 4. Binding and cleavage of any substrate by Fpg.
Figure 1
Figure 1
Structures of DNA lesions used in this work. 8-OxoG, 8-oxo-2′-deoxyguanosine; AP, abasic site; F, tetrahydrofuran abasic site analogue.
Figure 2
Figure 2
Stopped-flow fluorescence traces obtained for a non-cleavable F-containing ligand. Coloured traces represent experimental data; black curves are theoretically fitted.
Figure 3
Figure 3
Fluorescence traces obtained for a natural (cleavable) AP-containing substrate. Experimental data and fitting results are colour-coded as in Figure 2.
Figure 4
Figure 4
Change in Fpg fluorescence during titration with product oligonucleotides. [Fpg] = 1.5 µM. Square, 12mer GGAAGGCGAGAG; circle, 6mer pCCTTCC; cross, 5mer CTCTCp; diamonds, mixture of 12mer + 6mer + 5mer. Concentrations of 12, 6 and 5mers in the first three experiments are adjusted to equal the total concentration of individual chains in the fourth experiment.
Figure 5
Figure 5
Fluorescence traces obtained for a cleavable 8-oxoG-containing substrate. Experimental data and fitting results are colour-coded as in Figure 2.
Figure 6
Figure 6
Tryptophan residues in the structure of Fpg. A cartoon representation of the protein is shown with DNA in yellow sticks. Side chains of tryptophan residues are shown in blue sticks and labelled.
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References

    1. von Sonntag C. (1987) The Chemical Basis of Radiation Biology. Taylor & Francis, London.
    1. Halliwell B. and Gutteridge,J.M.C. (1999) Free Radicals in Biology and Medicine, 3rd Edn. Oxford University Press, Oxford, UK.
    1. Shibutani S., Takeshita,M. and Grollman,A.P. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature, 349, 431–434. - PubMed
    1. Moriya M., Ou,C., Bodepudi,V., Johnson,F., Takeshita,M. and Grollman,A.P. (1991) Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat. Res., 254, 281–288. - PubMed
    1. Moriya M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G·C→T·A transversions in simian kidney cells. Proc. Natl Acad. Sci. USA, 90, 1122–1126. - PMC - PubMed

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