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Kinetic Conformational Analysis of Human 8-Oxoguanine-DNA Glycosylase

2006; Elsevier BV; Volume: 282; Issue: 2 Linguagem: Inglês

10.1074/jbc.m605788200

1083-351X

Nikita A. Kuznetsov, Vladimir V. Koval, Georgy A. Nevinsky, Kenneth T. Douglas, Dmitry O. Zharkov, Olga S. Fedorova,

Cancer therapeutics and mechanisms

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower β-elimination reaction at the 3′-side of the resulting abasic site. Many features of OGG1 mechanism, including its low β-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of β-elimination rather than pre-excision dynamics of DNA and the enzyme. 7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower β-elimination reaction at the 3′-side of the resulting abasic site. Many features of OGG1 mechanism, including its low β-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of β-elimination rather than pre-excision dynamics of DNA and the enzyme. Living cells continuously experience a great number of insults from reactive oxygen species that are both produced during aerobic respiration and generated by environmental factors such as ionizing radiation (1Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine.2nd Ed. Clarendon Press, Oxford1989Google Scholar). The adverse effects of oxidative damage to DNA include miscoding and mutagenesis, cytotoxicity, and disregulation of gene expression and may ultimately lead to cancers and aging (2Beckman K.B. Ames B.N. Physiol. Rev. 1998; 78: 547-581Crossref PubMed Scopus (3156) Google Scholar). To counteract these effects, cells maintain an extensive system of antioxidant defense, one branch of which is formed by DNA repair mechanisms (3Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.2006Google Scholar). Base excision repair is one subpathway that mostly deals with non-bulky base lesions and single-strand breaks, the lesions that are predominantly produced by oxidative damage (4Burrows C.J. Muller J.G. Chem. Rev. 1998; 98: 1109-1151Crossref PubMed Scopus (1627) Google Scholar, 5Pogozelski W.K. Tullius T.D. Chem. Rev. 1998; 98: 1089-1107Crossref PubMed Scopus (989) Google Scholar). Base excision repair of damaged bases is initiated by DNA glycosylases, enzymes that recognize such lesions and hydrolyze their N-glycosidic bonds (3Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.2006Google Scholar). In eukaryotes, a prominent role is played by 8-oxoguanine-DNA glycosylase OGG1, which excises 8-oxoguanine (8-oxoG) 3The abbreviations used are: 8-oxoG, 7,8-dihydro-8-oxoguanine; 8-BrG, 8-bromoguanine; AP, apurinic/apyrimidinic; 2-aPu, 2-aminopurine; F, 3-hydroxytetrahydrofuran-2-yl)methylphosphate (tetrahydrofuran abasic site analog); HPLC, high pressure liquid chromatography; ODN, oligodeoxynucleotide. (Fig. 1), a major damaged purine, from DNA (6Auffret van der Kemp P. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (348) Google Scholar, 7Radicella J.P. Dherin C. Desmaze C. Fox M.S. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8010-8015Crossref PubMed Scopus (571) Google Scholar, 8Rosenquist T.A. Zharkov D.O. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7429-7434Crossref PubMed Scopus (459) Google Scholar). Defects in OGG1 have been associated with human cancers (9Weiss J.M. Goode E.L. Ladiges W.C. Ulrich C.M. Mol. Carcinog. 2005; 42: 127-141Crossref PubMed Scopus (225) Google Scholar) and enhanced mutagenesis (10Thomas D. Scot A.D. Barbey R. Padula M. Boiteux S. Mol. Gen. Genet. 1997; 254: 171-178Crossref PubMed Scopus (203) Google Scholar, 11Arai T. Kelly V.P. Komoro K. Minowa O. Noda T. Nishimura S. Cancer Res. 2003; 63: 4287-4292PubMed Google Scholar), and accelerated senescence has been observed in a mouse strain with thermolabile OGG1 (12Choi J.-Y. Kim H.-S. Kang H.-K. Lee D.-W. Choi E.-M. Chung M.-H. Free Radic. Biol. Med. 1999; 27: 848-854Crossref PubMed Scopus (44) Google Scholar). In addition to 8-oxoG, OGG1 has been shown to excise formamidopyrimidine derivatives of G (13Karahalil B. Girard P.-M. Boiteux S. Dizdaroglu M. Nucleic Acids Res. 1998; 26: 1228-1232Crossref PubMed Scopus (109) Google Scholar, 14Dherin C. Radicella J.P. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1999; 27: 4001-4007Crossref PubMed Scopus (244) Google Scholar). During the reaction, a covalent Schiff base intermediate is formed between an active site lysine residue (Lys-249 in human OGG1) and C-1′ of the damaged nucleoside (15Nash H.M. Lu R. Lane W.S. Verdine G.L. Chem. Biol. 1997; 4: 693-702Abstract Full Text PDF PubMed Scopus (162) Google Scholar). Similar to other DNA glycosylases that form the Schiff base (16Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar), OGG1 possesses an abasic (apurinic/apyrimidinic (AP)) lyase activity, which catalyzes β-elimination at the nascent AP site to break the damaged DNA strand. This activity is weak, being about 1 order of magnitude lower than the glycosylase activity (17Bjørås M. Luna L. Johnsen B. Hoff E. Haug T. Rognes T. Seeberg E. EMBO J. 1997; 16: 6314-6322Crossref PubMed Scopus (331) Google Scholar, 18Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), but can be enhanced by analogs of 8-oxoG such as 8-bromoguanine (8-BrG) (19Fromme J.C. Bruner S.D. Yang W. Karplus M. Verdine G.L. Nat. Struct. Biol. 2003; 10: 204-211Crossref PubMed Scopus (146) Google Scholar, 20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar). OGG1 demonstrates a profound specificity for the base opposite the lesion, especially if DNA strand cleavage is used as the assay end point, with C being preferred at this position and purines strongly discriminated against (17Bjørås M. Luna L. Johnsen B. Hoff E. Haug T. Rognes T. Seeberg E. EMBO J. 1997; 16: 6314-6322Crossref PubMed Scopus (331) Google Scholar, 18Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). If C is opposite the lesion, the enzyme may even excise bases not cleaved in their natural context; for example, it excises 8-oxoadenine from 8-oxoA:C mispairs (18Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 21Girard P.M. D'Ham C. Cadet J. Boiteux S. Carcinogenesis. 1998; 19: 1299-1305Crossref PubMed Scopus (74) Google Scholar). As with many other DNA glycosylases, the structure of OGG1 is known (22Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar), but not all aspects of its substrate preference can be explained solely by structural considerations, suggesting that dynamic features of lesion recognition can contribute to the enzyme specificity (reviewed in Ref. 23Zharkov D.O. Grollman A.P. Mutat. Res. 2005; 577: 24-54Crossref PubMed Scopus (75) Google Scholar). Recognition and excision of damaged bases by DNA glycosylases is accompanied by several conformational rearrangements that bring the base into the enzyme catalytic site. As a rule, DNA is severely kinked at the site of the lesion, the damaged nucleotide is everted from the double helix and inserted in a deep catalytic pocket, and several amino acid residues of the enzyme are inserted into the resulting void in DNA (a process commonly referred to as "plugging") to stabilize the whole structure (for a recent review, see Ref. 24Huffman J.L. Sundheim O. Tainer J.A. Mutat. Res. 2005; 577: 55-76Crossref PubMed Scopus (185) Google Scholar). These processes occur in a rapid consequence and are not easily studied by conventional steady-state enzyme kinetic methods. Recently, stopped-flow techniques have been applied by several groups, including ours, to investigate the multiple conformational changes accompanying damage recognition (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar, 25Jiang Y.L. Stivers J.T. Biochemistry. 2002; 41: 11236-11247Crossref PubMed Scopus (57) Google Scholar, 26Wong I. Lundquist A.J. Bernards A.S. Mosbaugh D.W. J. Biol. Chem. 2002; 277: 19424-19432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 27Wong I. Bernards A.S. Miller J.K. Wirz J.A. J. Biol. Chem. 2003; 278: 2411-2418Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 28Fedorova O.S. Nevinsky G.A. Koval V.V. Ishchenko A.A. Vasilenko N.L. Douglas K.T. Biochemistry. 2002; 41: 1520-1528Crossref PubMed Scopus (59) Google Scholar, 29Koval V.V. Kuznetsov N.A. Zharkov D.O. Ishchenko A.A. Douglas K.T. Nevinsky G.A. Fedorova O.S. Nucleic Acids Res. 2004; 32: 926-935Crossref PubMed Scopus (58) Google Scholar). In particular, we have used stopped-flow with detection of intrinsic tryptophan (Trp) fluorescence to study the dynamics of 8-oxoG recognition and removal by human OGG1 and its activation by 8-BrG (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar). We were able to derive a minimal kinetic scheme (Scheme I) of this process and to suggest the conformational changes underlying each step. However, DNA conformational dynamics during OGG1 catalysis was not addressed, and neither was the amazing opposite-base specificity of this enzyme. In the present study, we have investigated changes in the conformation of DNA processed by OGG1 by using 2-aminopurine (2-aPu) as a fluorescent marker complementing the Trp fluorescent studies. This nucleobase analog has a high quantum yield in aqueous solution, but the fluorescence is highly quenched when it is incorporated into DNA or transferred into a nonpolar environment (30Ward D.C. Reich E. Stryer L. J. Biol. Chem. 1969; 244: 1228-1237Abstract Full Text PDF PubMed Google Scholar, 31Rachofsky E.L. Osman R. Ross J.B.A. Biochemistry. 2001; 40: 946-956Crossref PubMed Scopus (312) Google Scholar). These properties make 2-aPu attractive for stopped-flow analysis of protein-DNA interaction; indeed, the combination of Trp and 2-aPu fluorescence has been used to dissect the kinetic pathway of damage recognition by the repair enzyme uracil-DNA glycosylase (32Stivers J.T. Pankiewicz K.W. Watanabe K.A. Biochemistry. 1999; 38: 952-963Crossref PubMed Scopus (193) Google Scholar). We have also analyzed the DNA dynamics associated with stimulation of the AP lyase activity of the enzyme by 8-BrG. Finally, we have addressed the origins of OGG1 specificity for the base opposite the lesion by stopped-flow kinetics. Materials and Buffers—The chemicals used were purchased mostly from Sigma-Aldrich. 8-Bromoguanine was synthesized according to a published procedure (33Long R.A. Robins R.K. Townsend L.B. J. Org. Chem. 1967; 32: 2751-2756Crossref PubMed Scopus (99) Google Scholar). T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). [γ-32P]ATP (>3000 Ci/mmol) was purchased from Radioisotope (Moscow). Unless indicated otherwise, all experiments were carried out at 25 °C in a reaction buffer containing 50 mm Tris-HCl (pH 7.5), 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, and 9% glycerol (v/v). Oligodeoxynucleotides and Enzymes—Human OGG1 was purified as described (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar). The final enzyme stock of 23.4 μm, as determined spectrophotometrically using the Gill-von Hippel algorithm (34Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar) (ϵ280 = 6.84 × 104 m–1 cm–1), was stored at –20 °C. The purified OGG1 had 65% of the active enzyme form as determined by borohydride trapping with 8-oxoG substrate as follows. The reaction mixture (10 μl) included 2 μm enzyme, 25 mm potassium phosphate (pH 6.8), 100 mm NaCl, 100 mm NaBH4, and varying amounts of oligonucleotide duplex containing an 8-oxoG residue opposite C. The samples were incubated for 1 h at room temperature and analyzed by 12% SDS-PAGE. The gel was stained with Coomassie Blue and quantified using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Silver Spring, MD). The concentration of active form of enzyme was taken into account in all experiments. The sequences of ODNs used in this work are listed in Table 1. The ODNs were synthesized on an ASM-700 synthesizer (Biosset, Novosibirsk, Russia) using phosphoramidites purchased from Glen Research (Sterling, VA) and purified by anion exchange HPLC on a Nucleosil 100-10 N(CH3)2 column followed by reverse-phase HPLC on a Nucleosil 100-10 C18 column (both columns from Macherey-Nagel, Düren, Germany). The purity of ODNs exceeded 98% as estimated by electrophoresis in 20% denaturing PAGE after staining with the Stains-All dye (Sigma-Aldrich). The concentration of the ODNs was determined from their absorbance at 260 nm. ODN duplexes were prepared by annealing modified and complementary strands taken at 1:1 molar ratio in the reaction buffer described under "Materials and Buffers."TABLE 1Sequences of oligodeoxynucleotides used in this workShorthandSequenceoxoGd (CTCTC (oxoG) CCTTCC)oxoG-aPud (CTCTC (oxoG) (2 — aPu) CTTCC)AP-aPud (CTCTC (AP) (2 — aPu) CTTCC)F-aPud (CTCTCF (2 — aPu) CTTCC)G-aPud (CTCTCG (2 — aPu) CTTCC)Cd (GGAAGGCGAGAG)Ad (GGAAGGAGAGAG)Gd (GGAAGGGGAGAG)Td (GGAAGGTGAGAG)CCGd (GGAAGCCGAGAG) Open table in a new tab Stopped-flow Fluorescence Measurements—Stopped-flow measurements with fluorescence detection were carried out essentially as described (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar, 35Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Biochemistry. 2006; (in press)PubMed Google Scholar) using a model SX.18MV stopped-flow spectrometer (Applied Photophysics). To detect intrinsic Trp fluorescence only, λex = 283 nm was used and λem > 320 nm was followed as transmitted by a Schott filter WG 320 (Schott, Mainz, Germany). If 2-aPu was present in the ODNs, λex = 310 nm was used to excite 2-aPu residues, and their emission was followed at λem > 370 nm (Corion filter LG-370); Trp fluorescence of the enzyme (λex = 283 nm) was detected in this case using a Corion filter P10-340, which transmits a 10-nm-wide band at ∼340 nm to avoid overlapping with 2-aPu emission. The dead time of the instrument was 1.4 ms. The concentration of OGG1 in all experiments with Trp fluorescence detection was 2 μm, and concentrations of ODN substrates were varied from 0.5 to 4 μm. The concentration of substrates containing 2-aPu in experiments with 2-aPu fluorescence detection was 1 μm, and concentrations of OGG1 protein were varied from 0.5 to 4 μm. Concentrations of reactants reported are those in the reaction chamber after mixing. Typically, each trace shown is the average of four or more individual experiments. Bleaching of Enzyme Fluorescence—For the correction of the measured data on bleaching effect, the fluorescence intensities were recalculated using Equation 1 (28Fedorova O.S. Nevinsky G.A. Koval V.V. Ishchenko A.A. Vasilenko N.L. Douglas K.T. Biochemistry. 2002; 41: 1520-1528Crossref PubMed Scopus (59) Google Scholar), F=(Fobs-Fb)×exp(kb×t)+Fb(Eq. 1) where F is the corrected fluorescence intensity, Fobs is the observed fluorescence intensity, Fb is the background fluorescence, and kb is the coefficient determined for each substrate concentration in experiments with noncleaved substrates. The difference between the observed and corrected values did not exceed 10%. Global Nonlinear Simulation Fitting of Stopped-flow Data—Accurate modeling of all stopped-flow traces was obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) (36Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1360) Google Scholar) as described (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar, 29Koval V.V. Kuznetsov N.A. Zharkov D.O. Ishchenko A.A. Douglas K.T. Nevinsky G.A. Fedorova O.S. Nucleic Acids Res. 2004; 32: 926-935Crossref PubMed Scopus (58) Google Scholar, 35Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Biochemistry. 2006; (in press)PubMed Google Scholar). Differential equations were written for each species in the mechanisms described by Schemes I, II, III, IV, V, VI, VII (see "Results"), and the stopped-flow fluorescence traces were directly fit by expressing the corrected fluorescence intensity (Fc) at any reaction time t as the sum of the background fluorescence (Fb) and the fluorescence intensities of each protein species, Fc=Fb+∑i=0nFi(t)(Eq. 2) where Fi(t) = fi(Ei(t)), fi are the coefficients of specific fluorescence for each discernible OGG1 conformer, and (Ei(t)) are the concentrations of the conformers at any given time t (i = 0 relates to the free protein and i > 0, to the protein-DNA complexes). These specific fluorescence coefficients describe only the part of fluorescence that changes due to DNA binding. The minimal nature of the reaction schemes was confirmed by analyzing the dependence of the standard deviation of the residuals on the number of steps in the scheme using a scree plot (35Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Biochemistry. 2006; (in press)PubMed Google Scholar).SCHEME IIIKinetic mechanism of OGG1 processing of the AP substrate. E, OGG1; AP, AP substrate; E·AP, enzyme-substrate complex; P, reaction product; EP, enzyme-product complex; k1, k–1, and k2, individual rate constants; Kp, dissociation constant of the enzyme-product complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SCHEME IVAccumulation of the AP intermediate in OGG1-catalyzed reaction. E, OGG1; OG, 8-oxoG substrate; E·OG, enzyme-substrate complex; AP, product of base excision; Kbind, equilibrium constant of E·OG formation; kglyc, rate constant of base excision.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SCHEME VAccumulation of the β-elimination product in OGG1-catalyzed reaction. E, OGG1; OG, 8-oxoG substrate; E·OG and E·AP, enzyme-substrate complexes; P, product of β-elimination; Kbind, equilibrium constant of E·OG formation; kglyc and kelim, rate constants of base excision and β-elimination reaction, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SCHEME VIKinetic mechanism of OGG1 processing of the 8-oxoG substrate in the presence of 8-BrG. E, OGG1; OG, 8-oxoG substrate; (E·OG)n and E·AP, different enzyme-substrate complexes; P, reaction product; E·P, enzyme-product complex; ki and k–i, individual rate constants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SCHEME VIIAccumulation of the β-elimination product in OGG1-catalyzed reaction in the presence of 8-BrG. E, OGG1; OG, 8-oxoG substrate; E·OG, enzyme-substrate complex; P, product of β-elimination; Kbind, equilibrium constant of E·OG formation; kelim, rate constant of β-elimination.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Product Analysis—To analyze products formed by OGG1, the substrate oligonucleotides were 5′-32P-labeled using T4 polynucleotide kinase and [γ-32P]ATP. Reaction mixtures (20 μl) contained reaction buffer, 1, 2, or 4 μm 32P-labeled substrate, and 2 μm enzyme. The reaction was initiated by adding the enzyme and allowed to proceed at 25 °C for 25–60 min. Aliquots (2 μl) were withdrawn as required, mixed with 3 μl of gel-loading dye containing 7 m urea, and analyzed by 20% denaturing PAGE. The gels were exposed to Agfa CP-BU x-ray film (Agfa-Geavert), and the autoradiograms were scanned and quantified using Gel-Pro Analyzer, version 4.0. Kinetic parameters were obtained by numerical fitting using Microcal Origin version 7.0 software (OriginLab, Northampton, MA). Recently, we studied the dynamics of fluorescence of OGG1 tryptophan residues in the course of enzymatic reaction using stopped-flow technique (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar). When the substrate contained 8-oxoG opposite C, a series of changes in Trp fluorescence intensity were observed, attributed to binding and catalytic stages of enzyme process. The proposed kinetic scheme of OGG1 interaction with its DNA substrate included at least three fluorescently discernible consecutive steps accompanying damaged base recognition and its binding in the active site of the enzyme. The rate of damaged base excision (DNA glycosylase activity) by OGG1 is ∼10-fold higher than the rate of elimination of the 3′-phosphate of the damaged nucleotide (AP lyase activity) (18Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). This difference allowed us to propose the kinetic mechanism containing three equilibrium steps of the specific complex formation and two non-equilibrium catalytic steps. The nonequilibrium steps were attributed to glycosylase reaction and β-elimination. At the end of the process, formation of an enzyme-product complex was observed indicated by a decrease in final Trp fluorescence intensity in comparison with its initial values (Scheme I). However, only weak changes in the fluorescence intensity of the Trp residues of the enzyme were detected when OGG1 interacted with duplexes containing G, F, or AP residues opposite C (20Kuznetsov N.A. Koval V.V. Zharkov D.O. Nevinsky G.A. Douglas K.T. Fedorova O.S. Nucleic Acids Res. 2005; 33: 3919-3931Crossref PubMed Scopus (93) Google Scholar). In the present work, to gain a better understanding of the recognition of various substrates by OGG1, we studied a set of model duplexes (see Table 1 for the sequences) in which the target residue (8-oxoG, AP site, F, or G; Fig. 1) was flanked by 2-aPu, a fluorescent marker sensitive to the structure of DNA duplex. This approach allowed us to observe conformational changes in the oligonucleotide duplex in parallel with those in the enzyme and to assign conformational changes in the interacting molecules to the elementary steps in Scheme I. Furthermore, we determined the enzyme specificity for substrates with different bases opposite 8-oxoG and defined the stages of discrimination of these substrates by OGG1. We also analyzed the effect of 8-BrG on the processing of substrates containing 8-oxoG or AP. G-ligand—The process of OGG1 binding the nonspecific G-ligand was essentially complete by 0.02 s (Fig. 2A). An increase in fluorescence intensity of 2-aPu was observed, indicating destabilization of Watson-Crick or stacking interactions in the primary nonspecific enzyme-DNA complex (30Ward D.C. Reich E. Stryer L. J. Biol. Chem. 1969; 244: 1228-1237Abstract Full Text PDF PubMed Google Scholar, 31Rachofsky E.L. Osman R. Ross J.B.A. Biochemistry. 2001; 40: 946-956Crossref PubMed Scopus (312) Google Scholar). Fitting the experimental data to the one-site binding model (Scheme II) gave the values for the forward and reverse rate constants, k1 = (2.4 ± 0.1) × 108 m–1 s–1 and k–1 = 24.2 ± 5.0 s–1, respectively. F-ligand—The kinetic curves for the F-ligand, a non-cleavable analog of the AP site, were characterized by a decrease in 2-aPu fluorescence intensity during 5 s, suggestive of transition of the 2-aPu residue to a more hydrophobic environment (Fig. 2B). This could be the result of filling the abasic void in the DNA duplex by amino acids of OGG1 (37Norman D.P.G. Bruner S.D. Verdine G.L. J. Am. Chem. Soc. 2001; 123: 359-360Crossref PubMed Scopus (76) Google Scholar). The rate constants of the forward and reverse reactions of this single-stage mechanism (Scheme II) are k1 = (0.48 ± 0.05) × 106 m–1 s–1 and k–1 = 0.23 ± 0.04 s–1, respectively. AP Substrate—The substrate containing the AP site is expected to interact with the enzyme in a more complicated way. In this case, DNA binding and void-filling by OGG1 should be followed by β-elimination and dissociation of the enzyme-product complex. The last process resulted in an increase in the 2-aPu fluorescence intensity due to a transition of the 2-aminopurine to a more hydrophilic environment (Fig. 2C) at times >100 s. Scheme III describes the observed fluorescence changes in minimal terms. Its first step obviously reflects substrate binding and the transition to the catalytically active complex (E·AP). The irreversible step was attributed to the reaction of β-elimination. Therefore, the final step of the scheme most likely corresponds to the equilibrium between OGG1 and the reaction product. However, although we did see the beginning of this last reversible stage, the equilibrium could not be achieved, likely because of a very tight product binding. Therefore, numerical values were not calculated for Kp; Table 2 presents the rate constants obtained by fitting. The forward and reverse rate constants of the binding step (k1 and k–1, respectively) were close for the F-ligand and AP substrate, suggesting that this step reflects identical processes in both cases.TABLE 2The rate constants for interactions of OGG1 with G- and F-ligands and AP and 8-oxoG substratesConstantsaKbind=∑i=1i=5∏j=1j=iKj.G2-aPuF2-aPuAP2-aPuoxoG2-aPuoxoGTrpk1 (M-1 × s-1)(2.4 ± 0.1) × 108(0.48 ± 0.05) × 106(0.34 ± 0.02) × 106(1.2 ± 0.1) × 108(2.0 ± 0.5) × 108k-1 (s-1)24 ± 50.23 ± 0.040.13 ± 0.03120 ± 10230 ± 90k2 (s-1)0.0015 ± 0.00021.4 ± 0.12.5 ± 0.4k-2 (s-1)1.5 ± 0.10.59 ± 0.07k3 (s-1)0.10 ± 0.010.035 ± 0.010k-3 (s-1)0.013 ± 0.0020.019 ± 0.003k4 (s-1)0.029 ± 0.0010.040 ± 0.001kglyc (s-1)0.03k5 (s-1)0.0036 ± 0.00020.0065 ± 0.0001kelim (s-1)0.006KP (M)NDbND, not determined.(7.0 ± 1.1) × 10-6(3.3 ± 0.4) × 10-6Kbind (M-1)1 × 1072.1 × 1062.6 × 1069.2 × 1061.2 × 107a Kbind=∑i=1i=5∏j=1j=iKj.b ND, not determined.