DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

DNA Damage Processing by Human 8-Oxoguanine-DNAGlycosylase Mutants with the Occluded Active Site*

Received for publication, May 22, 2013, and in revised form, August 15, 2013 Published, JBC Papers in Press, August 17, 2013, DOI 10.1074/jbc.M113.487322

Maria V. Lukina‡§1, Alexander V. Popov‡1, Vladimir V. Koval‡§, Yuri N. Vorobjev‡§, Olga S. Fedorova‡§2,and Dmitry O. Zharkov‡§3

From the ‡Siberian Branch of the Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine,8 Lavrentieva Avenue, Novosibirsk 630090 and the §Department of Molecular Biology, Faculty of Natural Sciences,Novosibirsk State University, 2 Pirogova Street, Novosibirsk 630090, Russia

Background:Oxoguanine-DNA glycosylase (OGG1) removes highly mutagenic 8-oxoguanine from DNA.Results:OGG1mutations C253I and C253L occlude the active site and distort theOGG1-DNAprecatalytic complex but retainsome activity.Conclusion: Active site of OGG1 possesses flexibility that partially compensates for distortions.Significance: Active site plasticity may be important for dynamic recognition of multiple DNA lesions by DNA glycosylases.

8-Oxoguanine-DNA glycosylase (OGG1) removes premuta-genic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicksthe nascent abasic (apurinic/apyrimidinic) site by �-elimina-tion. Although the structure of OGG1 bound to damaged DNAis known, the dynamic aspects of 8-oxo-G recognition are notwell understood. To comprehend the mechanisms of substraterecognition and processing, we have constructed OGG1mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulkyleucine or isoleucine. The conformational dynamics of OGG1mutants were characterized by single-turnover kinetics andstopped-flow kinetics with fluorescent detection. Additionally,the conformational mobility of wild type and the mutant OGG1substrate complex was assessed using molecular dynamics sim-ulations.Althoughpocket occlusiondistorted the active site andgreatly decreased the catalytic activity of OGG1, it did not fullyprevent processing of 8-oxo-G and apurinic/apyrimidinic sites.Both mutants were notably stimulated in the presence of free8-bromoguanine, indicating that this base can bind to the dis-torted OGG1 and facilitate �-elimination. The results agreewith the concept of enzyme plasticity, suggesting that the activesite of OGG1 is flexible enough to compensate partially for dis-tortions caused by mutation.

8-Oxoguanine-DNA glycosylase (OGG1)4 is a eukaryoticDNA repair enzyme that removes 8-oxoguanine from DNA

(1–3). 8-Oxoguanine (8-oxo-G), a premutagenic oxidativepurine base lesion easily generated under oxidative stress con-ditions (4, 5), directs preferential incorporation of dAMP byDNA polymerases and thus produces G3T transversions aftertwo rounds of replication (6, 7). Therefore, cellular systems for8-oxo-G repair should quickly excise this base from pairs withC while limiting its activity on 8-oxo-G:A pairs, otherwise theG3T transversion would occur immediately (7).

The structure of human OGG1 has been determined for thefree enzyme and a number of its complexes with DNA approxi-mating various points along the reaction coordinate (8–19).OGG1belongs to the endonuclease III structural superfamily (20),possessing a characteristic helix-hairpin-helix motif and anextended loop rich in Gly and Pro and containing an absolutelyconserved catalytic Asp-268 residue. Another absolutely con-served residue, Lys-249, acts as a nucleophile that attacks C1� ofthe damaged nucleotide during the reaction and forms a transientcovalent intermediate, the Schiff base (21). The enzyme features awide positively charged groove where DNA binds. Upon binding,DNA is kinked by�70°, and the 8-oxo-G base is everted from thedouble helix. A narrow deep pocket within the DNA bindinggroove accommodates the damaged base, whereas the void left inDNA after the eversion is filled by Asn-149 and Asn-150.In the base-binding pocket, the flipped out 8-oxo-G is tightly

sandwiched between the aromatic system of Phe-319 and thethiol group of Cys-253, which, as quantummechanical calcula-tions show (14), likely exists as a deprotonated thiolate anion,stabilized by interactions with the positively charged Lys-249.The edges of the 8-oxo-G base form several hydrogen bondswith Gly-42, Asp-168, and Gln-315 (Fig. 1A).In addition to its ability to excise 8-oxo-G, OGG1 possesses a

slower activity that nicks DNA by the �-elimination mecha-nism (AP lyase activity). The DNA substrates for this activitymay contain either 8-oxo-G or preformed AP sites. In the lattercase, the structure of OGG1 bound to DNA containing anuncleavable AP site analog (3-hydroxytetrahydrofuran-2-

* This work was supported, in whole or in part, by National Institutes of HealthGrant CA017395 from NCI. This work was also supported by Presidium ofthe Russian Academy of Sciences Grants 6.11 and 6.12, Russian Foundationfor Basic Research Grants 11-04-00807, 12-04-00135, and 13-04-00013,Russian Ministry of Education and Science Grants SS-64.2012.4, 8092, and8473, Russian Government Grant to Leading Scientists 11.G34.31.0045,and funds from the Siberian Branch of the Russian Academy of Sciences.

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed. Tel.: 7-383-3635175; Fax:

7-383-3635153; E-mail: [email protected] To whom correspondence may be addressed. Tel.: 7-383-3635128; Fax:

7-383-3635153; E-mail: [email protected] The abbreviations used are: OGG1, 8-oxoguanine-DNA glycosylase, AP,

apurinic/apyrimidinic; BrG, 8-bromoguanine; ODN, oligodeoxyribonucleo-tide; 8-oxo-G, 8-oxoguanine; PDB, Protein Data Bank; r.m.s.d., rootmean square deviation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 40, pp. 28936 –28947, October 4, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

28936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 40 • OCTOBER 4, 2013

yl)methyl phosphate shows that the base-binding pocket remainsempty despite that the sugar moiety is everted in much the sameway as the damaged base.To understand the importance of the interactions within the

base-binding pocket for different reactions catalyzed byOGG1ondifferent substrates, we have constructed site-directed mutantsreplacing the Cys-253 residue with either leucine or isoleucine(Fig. 1A). Thehypothesiswas that the added steric bulk and inabil-ity to form the Cys-253(thiolate)–Lys-249 dipole would exclude8-oxo-G from the pocket and disable its excision but still allow theenzyme to act onAP sites.Wehave characterized the reactions bythese active site occlusionmutants using high resolution stopped-flow kineticswith fluorescence detection andmolecular dynamicssimulation. Overall, our results suggest that the mutations distortthe active site to the disadvantage of the catalytic step of the reac-tion,whereas the recognitionof 8-oxo-G is still efficient.However,the active site of OGG1 appears to be flexible enough to retainsome residual repair activity.

EXPERIMENTAL PROCEDURES

Oligonucleotide Synthesis and Purification—Oligodeoxyri-bonucleotides (ODNs; Fig. 1) were synthesized on an ASM-700synthesizer (BIOSSET, Novosibirsk, Russia) using phosphor-amidites purchased from Glen Research (Sterling, VA) andpurified by anion exchange high performance liquid chromato-graphy (HPLC) on a Nucleosil 100–10 N(CH3)2 column fol-lowed by reverse-phase HPLC on a Nucleosil 100-10 C18 col-umn (both columns from Macherey-Nagel, Duren, Germany).The purity of the ODNs was assessed by 20% denaturing PAGEafter staining with Stains-All (Sigma). The concentrations ofthe ODNs were determined from their absorbance at 260 nm.The AP-containing ODN was prepared by incubating 0.1

mmol of the ODN containing dU at the intended place of theAP site for 14 h at 37 °Cwith 15 units of uracil-DNAglycosylase(New England Biolabs, Ipswich, MA) in 150 �l of the buffer

containing 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithio-threitol (DTT), and 0.1 mg/ml bovine serum albumin. Thereaction product was purified by reverse-phase HPLC on aNucleosil 100-5 C18 column using a linear gradient of 0–20% ace-tonitrile in 0.1 M triethylammonium acetate (pH 7.0). The pooledfractionswere concentrated and then converted to the lithiumsaltform using a Sep-Pak Plus C18 cartridge (Waters, Milford, MA).The integrity of the AP-containing ODN was assessed by PAGEfollowedby Stains-All staining. To confirm the presence of theAPsite in the ODN after the treatment with uracil-DNA glycosylase,samples were treated with 10% aqueous piperidine at 95 °C andwere completely cleaved at the modified site. When needed, themodifiedstrandswere 32P-labeledusing [�-32P]ATPandphageT4polynucleotide kinase (SibEnzyme, Novosibirsk, Russia) accord-ing to the manufacturer’s protocol, purified by 20% denaturingPAGE, and annealed to the complementary strand.Enzymes—Site-directed mutants were constructed using a

QuikChangemultisite-directedmutagenesis kit (Agilent Tech-nologies, Santa Clara, CA); pET-15b plasmid carrying theOGG1 isoform 1a insert (22) served as a template The mutantproteins were induced and purified as described for the wild-type OGG1 (22). As the mutants proved to retain their activityon the AP substrates, concentration of the active form wasdetermined from burst phase kinetics using the AP substrate asdescribed (23).Stopped-flow Measurements—Stopped-flow measurements

with fluorescence detection were carried out using a modelSX.18MV stopped-flow spectrometer (Applied Photophysics,Leatherhead, UK) fitted with a 150-watt xenon arc lamp. Allexperiments were carried out at 25 °C in a buffer containing 50mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM DTT,and 9% (v/v) glycerol. The enzyme in one syringe was rapidlymixed with the substrate solution in the other syringe. Thesubstrate and enzyme solutions were equilibrated for 45 min at

FIGURE 1. A, impact of the occluding mutations on the structure of OGG1. The 8-oxo-G nucleotide is shown as a van der Waals sphere model, and amino acidresidues Gln-315 and Phe-319 forming the 8-oxo-G-binding pocket are shown as ball-and-stick models colored according to the atom type (cyan, carbons; blue,nitrogens; red, oxygens; brown, phosphorus atoms). The amino acid residue at position 253 is color-coded: yellow, Cys-253 (wild type); cyan, Ile-253; magenta,Leu-253. The rest of the protein is shown as a semitransparent schematic model. The structure was built by direct replacement of Cys-253 using the mutagen-esis tool of PyMOL (47). B and C, sequence of oligodeoxyribonucleotides and chemical nature of the lesions (X) used in the kinetic experiments (B, AP site; C,8-oxo-2�-deoxyguanosine).

Damage Processing by OGG1 with the Occluded Active Site

OCTOBER 4, 2013 • VOLUME 288 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 28937

25 °C beforemixing. The concentrations aftermixing were var-ied in the 0.5–2 �M range for the ODN duplexes, and the con-centration of OGG1 in all experiments was 1 �M. Excitationwavelength �ex � 283 nm was used, and the fluorescent emis-sion was monitored using a 320-nm long pass filter (WG-320,Schott, Mainz, Germany) to detect the fluorescence of the pro-tein’s Trp residues. The dead time of the instrumentwas 1.4ms.Each trace obtained is the average of at least four individualexperiments.Data Processing—The approach is based on the fluorescence

intensity changes in the course of the reaction due to the for-mation of the enzyme-DNA complex and its subsequent trans-formation to conformers corresponding to different intermedi-ates of the substrate recognition and processing (22, 24, 25).The kinetic parameters were obtained by numerical integrationof the system of kinetic differential equations (Equation 1) andthe least squares global nonlinear fitting of the total fluores-cence (F, total fluorescence; Fb, background fluorescence; fi,fluorescence coefficients of individual OGG1 conformers; [Ei],concentrations of individual OGG1 conformers, where i � 0corresponds to the free enzyme) using the DynaFit package(BioKin, Pullman, WA) (26).

d�E�

dt� ��k1�S� � k�5�P��E� � k�1�ES1� � k5�EP�

d�S�

dt� �k1�E��S� � k�1�ES1�

d�ES1�

dt� k1�E��S� � �k�1 � k2��ES1� � k�2�ES2�

d�ES2�

dt� k2�ES1� � �k�2 � k3��ES2� � k�3�ES3�

d�ES3�

dt� k3�ES2� � �k�3 � k4��ES3�

d�EP�

dt� k4�ES3� � k5�EP� � k�5�E��P�

d�P�

dt� k5�EP� � k�5�E��P�

F � Fb � �i � 0

n

fi�Ei� (Eq. 1)

In the evaluated mechanisms, except for the first bimolecularstep and the product release step, all other reactions were firstorder. The kinetic parameters were obtained by global fitting ofsets of fluorescence curves obtained at different concentrationsof the reactants. During the fitting procedure, all relevant rateconstants for the forward and reverse reactions as well as thespecific molar response factors for all intermediate complexeswere optimized. The minimal kinetic mechanism (Scheme 1)was confirmed using a scree test as described for the wild-typeOGG1 (24).

OGG1Cleavage Assay and Product Accumulation Kinetics—To analyze the products formed by OGG1, the reaction wasperformed under the same conditions as the stopped-flowexperiments except the substrate ODNs were 32P-labeled. Ifneeded, the reaction mixture also contained 0.5 mM 8-bromo-guanine. The reaction was terminated at required time pointsby adding the loading dye solution containing 7 M urea. Todetermine the rate of DNA nicking (the AP lyase activity), thealiquots were directly analyzed by 20% denaturing PAGE. Toanalyze the rate of AP site formation from the substrate con-taining 8-oxo-G (theN-glycosylase activity), 10–12 volumes of2% LiClO4 in acetone were added to the aliquots, and the pre-cipitates were washed with 100 �l of 85% ethanol, then twicewith 100 �l of acetone, dried, and treated with 10% aqueouspiperidine at 95 °C for 30 min to cleave all AP sites introducedby the enzyme. The products were again precipitated, washed,and dried as describe above. The samples were dissolved in 3 �lof the loading dye solution and analyzed by 20% denaturingPAGE. The gels were exposed to Agfa CP-BU x-ray film (Agfa-Geavert, Mortsel, Belgium); the autoradiograms were scannedand quantified by scanning densitometry using Gel-Pro Ana-lyzer software (Media Cybernetics, Rockville, MD). The rateconstants were obtained by numerical integration of systems ofmass balance and kinetic differential equations assuming a fastequilibrium single-turnover model (Scheme 2, Equation 2; seeunder “Results” for details of the kinetic model choice) andfitting the product accumulation curves to the solution usingDynaFit.

For Scheme 2A

�E � DNAoxoG� � Ka�E��DNAoxoG�

d�E � DNAAP�

dt� kglyc�E � DNAoxoG�

For Scheme 2B

�E � DNAAP� � Ka�E��DNAAP�

d�E � DNAnick�

dt� kly�E � DNAAP�

For Scheme 2C

�E � DNAoxoG� � Ka�E��DNAoxoG�

d�E � DNAoxoG�

dt� �kglyc�E � DNAoxoG�

d�E � DNAAP�

dt� kglyc�E � DNAoxoG� � kly�E � DNAAP�

d�E � DNAnick�

dt� �kly�E � DNAAP� (Eq. 2)

SCHEME 1. Kinetic mechanism of interaction of OGG1 C253I and OGG1C253L with the 8-oxo-G substrate derived from the stopped-flow data.



where [E] is the concentration of the free enzyme; [DNAoxoG]and [DNAAP] are the concentrations of free 8-oxo-G and APsite-containing DNA, respectively; [E�DNAoxoG], [E�DNAAP],and [E�DNAnick] are the concentrations of complexes of theenzyme with 8-oxo-G, AP site-containing, and nicked DNA,respectively;Ka is the association constant; kglyc is the rate con-stant of base excision (the glycosylase reaction), and kly is therate constant of strand nicking (the AP lyase reaction).Molecular Dynamic Simulations—The atomic structure of

human OGG1 (PDB code 1EBM) containing a 15-mer-DNAduplex was taken as a starting structure for modeling (see Fig.5A for the sequence and numeration of the duplex). TheK249Qmutation, present in the structure to inactivate the enzyme,wasconverted back to Lys-249 by manually editing the PDB file.The residues 80–82 missing in the 1EBM structure wererestored using the following protocol. The initial backboneconformation of the residues 80–82 was taken from the freeOGG1 structure (PDB entry 1KO9). The fragment was alignedfor the best fit to the source structure and incorporated into thePDB file. Then the side chains of residues 80–82were restored,and their orientation was optimized in 500 steps of Fletcherenergy optimization algorithm. The final refinement was doneby 500 ps of simulated annealing using the GUI-BioPASEDmolecular modeling suite (27). It should be noted that residues80–82 reside on the protein surface far from the active site. Theobtained structure (model WT) contains cytosine opposite thelesion. The C253I and C253L mutant models were manuallyconstructed by side-chain replacement and adjustment of8-oxo-G base position, followed by energy optimization andmolecular dynamics-simulated annealing. The mutant struc-tures were finally validated using the PDB validation module ofGUI-BioPASED (27). Following the results of quantummechanics/molecular mechanics data (14) and OGG1 mecha-nism considerations (12), some amino acid residues were sim-ulated in nonstandard protonation state to approximate thesituation immediately preceding the nucleophilic attack. Cys-

253 was modeled in the deprotonated state as the thiolateanion, and Lys-249 and Asp-268 were modeled with neutralamino group and carboxyl group, respectively. The force fieldparameters of these charge states were derived from AMBERff99. The force field parameters for the 8-oxo-2�-deoxyguanosine-5�-phosphate residue were taken from Ref. 28. A 10-nsmolecular dynamics simulation was performed using theBioPASED molecular dynamics modeling software (29) usingtheAMBER ff99 force field and the analytical Gaussian solvent-exclusion implicit solvent model (30), with an integration timestep of 1 fs. The system was gradually heated from 10 to 300 Kover 50 ps and equilibrated at 300 K. A classic moleculardynamics trajectory was generated in the NTV ensemble withharmonic restraints of 0.001 kcal/A2 for the protein atoms, 0.25kcal/A2 for atoms of the terminal nucleotides, and 0.0025kcal/A2 for the rest of the DNA atoms. Coordinates of eachatom of the system (snapshots) were saved every 2 ps, thusproducing a trajectory of 5076 files. All trajectories were ana-lyzed using MDTRA trajectory analysis software (31).

RESULTS

Stopped-flowKinetics of C253I andC253LOGG1—Theover-all shape of Trp fluorescence traces observed for the interactionbetween C253I and C253L OGG1 and oxo-G substrate (Fig. 2)was quite similar to each other and to the Trp traces for wild-typeOGG1processing the same substrate (22, 24), yet the char-acteristic times were different. The fluorescence quicklydropped until �1 s. This step was followed by a much moregradual fluorescent signal decrease with a less pronouncedchange in the amplitude, and then the fluorescence started togrow at �200–300 s, but this process was still not complete by3000–4000 s. The time scale of the slow fluorescent changes isshifted toward longer times compared with wild-type OGG1.The initial fast decrease in the fluorescent signal can be associ-ated with DNA-enzyme complex formation, and no notabledifferences in fluorescent traces for mutant and wild-type

FIGURE 2. Fluorescent traces of the time course of interaction between 1 �M OGG1 C253L (A) or C253I (B) with the 8-oxo-G substrate at the indicatedconcentration. Jagged lines, fluorescent traces; smooth lines, fitted reaction progress curves.

SCHEME 2. Accumulation of abasic and nicked products.



OGG1 were detected at this step. According to the PAGE anal-yses discussed below, at this time scale (up to 4000 s) no nickedproduct was formed; therefore, the observed growth of fluores-cence probably reflects the abasic product release from theenzyme-substrate complex.Processing of the 8-oxo-G substrate by both mutant forms

was described using minimal Scheme 1, which was the same asfor thewild-typeOGG1 (22). Values for the individual rate con-stants were determined by global fitting (Table 1). Notably, k4,the rate constant of the irreversible step, was 15-fold lower forboth mutant forms in comparison with the wild-type enzyme(0.0046 and 0.0042 s�1 for OGG1 C253L and OGG1 C253I,respectively, in contrast to 0.06 s�1 for wild-type OGG1) (22).According to the PAGE analysis of C253I and C253L enzymes,the k4 rate constant is likely related to the AP site product for-mation, which is followed byDNA release; the same attributionof this reaction step was made earlier for wild-type OGG1 (22).In contrast to the catalytic step, the preceding stages of the

reaction, namely binding and structural adjustment of theenzyme-substrate complex, are affected to a lesser degree. Thegeneral association constant Ka, calculated using Equation 3,was 1.9 107 and 1.6 107 M�1 for OGG1 C253I and OGG1C253L, respectively, only �1.5-fold lower than for the wild-type enzyme (Ka � 2.9 107 M�1) (22). Thus, it seems likelythat bothmutations predominantly affect the catalytic ability ofOGG1 rather than its affinity for DNA or its capacity to distortDNA toward the precatalytic complex.

Ka �k1

k�1�

k1k2

k�1k�2�

k1k2k3

k�1k�2k�3(Eq. 3)

C253I andC253L Product Formation Rates—Presteady-statestopped-flow analysis with fluorescence detection permits thedetection of conformational changes in the enzyme molecule.To associate the conformational transitions with particularchemical steps of the catalytic cycle, we used PAGE to detectaccumulation of abasic and nicked products for C253I andC253L OGG1 (Fig. 3). No considerable difference in the prod-uct formation rates between the two mutant forms was regis-tered. However, in theOGG1mutant forms bothN-glycosylaseand AP lyase activities were significantly retarded comparedwith thewild-typeOGG1.With the 8-oxo-G substrate, the aba-sic product accumulation rates were �180- and �90-foldslower for C253I and C253L OGG1, respectively, than for thewild-type enzyme under the same conditions when OGG1 and8-oxo-G substratewere both taken at 1�M (Fig. 3A). The rate ofaccumulation of the nicked product for the 8-oxo-G substratewas affected by the mutations to a lesser degree, decreasing

4–8-fold only (Fig. 3B). For the AP substrate, as expected fromthe relaxed requirement for the base-binding pocket, thenicked product accumulation was faster than for the 8-oxo-Gsubstrate for both C253I and C253L OGG1 (Fig. 3B).

Rate constants or product formations were estimated fromthe PAGE analysis results. Because the concentrations of boththe enzyme and the substrate (1�M) greatly exceededKd values(�53 and 63 nM for C253I andOGG1C253L;Kdwas calculatedas 1/Ka, where Ka was from Equation 3) and the catalytic ratewas apparently low, the single-turnover model was used, andthe observed rate constant was taken as an approximation forthe rate constant of the catalytic step. The N-glycosylase reac-tion with the 8-oxo-G substrate and the AP lyase reaction withAP substrate were fitted as one irreversible step (Scheme 2, Aand B, respectively), whereas the combined N-glycosylase andAP lyase reactions with 8-oxo-G substrate were fitted as twosequential irreversible steps (Scheme 2C), and the rate constantof AP lyase reaction for 8-oxo-G substrate was calculated usingthe kglyc constant obtained for Scheme 2A. The calculated rateconstants are listed in Table 2. The apparent values of kglyc andkly for both C253I and C253L OGG1 were at least 1 order ofmagnitude lower than for wild-type OGG1 (22). The observed

TABLE 1Rate constants for interaction of OGG1 C253L and OGG1 C253I with the oxoG substrate calculated from the stopped-flow data

Rate constant Wild-type OGG1a OGG1 C253L OGG1 C253I

k1 (M�1 s�1) (260 10) 106 (28.5 1.3) 106 (13.8 0.6) 106k–1 (s�1) 130 1 5.7 0.6 3.3 0.3k2 (s�1) 13.3 0.2 3.1 0.3 3.5 0.4k–2 (s�1) 1.16 0.02 5.2 0.2 2.9 0.2k3 (s�1) 0.012 0.001 0.0116 0.0005 0.0163 0.0006k–3 (s�1) 0.07 0.01 0.0045 0.0003 0.0082 0.0004k4 (s�1) 0.06 0.02 0.0042 0.0003 0.0046 0.0002KP (M) 8.8 10�7 (1.1 0.1) 10�7 (1.3 0.1) 10�7

a Data are from Ref. 22.

FIGURE 3. Time course of product accumulation in the reaction catalyzedby wild-type (circles), C253I (squares), and C253L (triangles) OGG1 at 1�M enzyme and substrate concentrations. A, representative gel illustratingcleavage of 8-oxo-G substrate by wild-type OGG1 (no piperidine treatment,nicked product formation reveals AP lyase activity). Arrowheads indicate thefollowing: S, substrate oligonucleotide, P, product of cleavage. B, abasic prod-uct formation from the 8-oxo-G substrate. C, nicked product formation fromthe 8-oxo-G substrate (black symbols) or the AP substrate (white symbols). Band C, lines show the fit of the data to the solution of the respective system ofdifferential equations (Equation 2 and Scheme 2). Each symbol is the mean ofthree independent experiments; S.E. did not exceed 20%, error bars are omit-ted for clarity.



kglyc constants were of the same order of magnitude as the k4constants determined from stopped-flow experiments butexceeded the kly constants 20–80-fold, indicating that in thestopped-flow experiments, the released product was indeed theAP site rather than nicked DNA. Notably, the kly constant forthe AP substrate was 2.8–4.4-fold higher than for the 8-oxo-Gsubstrate, again consistent with the idea that the AP site doesnot enter the base-binding pocket deeply, and therefore itscleavage should be less affected by the active site occlusion.Effect of 8-Bromoguanine on the Interaction of OGG1 with

8-Oxoguanine and AP Substrates—We and others have previ-ously shown that the AP lyase reaction catalyzed by OGG1 isstimulated by adding free 8-bromoguanine base (BrG) to thereaction mixture (12, 22, 24). The effect of BrG on the AP lyaseactivity of C253I and C253L OGG1 was studied using PAGEanalysis (Fig. 4). For both mutant forms, a notable increase inthe nicked product formation rates was observed during inter-action with both AP- and 8-oxo-G substrates in the presence of0.5 mM BrG. In the case of the AP substrate (Fig. 4, A and C),BrG enhanced the reaction rate 5–15-fold for both mutants andwild-type OGG1. The stimulation of strand nicking was observedbothunder the single-turnover (Fig. 4A) and for themultiple turn-over conditions (Fig. 4C).With the8-oxo-Gsubstrate, two typesofexperiments were conducted (Fig. 4B). In one setup, BrG wasadded to the reactionmixture a few seconds after the protein, andthe substrates were combined. In the other setup, OGG1was pre-mixedwithBrGfor45minbefore thesubstratewas introduced.Aswith the AP substrate, a significant acceleration of the AP lyasereaction was observed for both C253I and C253L mutant forms.

Of note, the �-elimination rate did not depend on the order ofmixing. This may indicate that even if BrG binds to the occludedOGG1 active site in the absence of DNA, this binding is not tightenough to completely prevent subsequent entrance and coordina-tion of 8-oxo-G substrate.Molecular Dynamics of OGG1 C253I and C253L—To gain

insight into the structural consequences of the Cys-253replacement, we have performed molecular dynamic studies ofwild-typeOGG1 and themutants bound toDNA.As the exper-iments suggested that the initial stages of DNA binding andlesion recognition are probably unaffected by the mutations,and they slow down the reaction by perturbing the Michaeliscomplex, our main goal was to probe the conformational spaceavailable to the enzyme with the base everted from DNA andpoised for excision. Therefore, we used a crystal structure ofcatalytically inactivated OGG1 bound to DNA with 8-oxo-Gfully in the lesion recognition pocket (1EBM in PDB) as a start-ingmodel and an implicit solvent system to accelerate the sam-pling of available conformations of the active site (32). Thedynamics of the active site can be affected by the charges ofionizable groups inside. In the OGG1 reaction intermediateimmediately preceding the nucleophilic attack at C1�, Lys-249has to be deprotonated to expose a lone electron pair for theattack, whereas Asp-268 is juxtaposed to a partially negativelycharged O4�, which receives a proton to open the deoxyribosering (12), so Asp-268 is likely protonated. Accordingly, wechose tomodel both Lys-249 andAsp-268 in the neutral state inall three structures. Furthermore, in wild-type OGG1, we havesimulated Cys-253 as a thiolate anion, following the quantum

FIGURE 4. Time course of product accumulation in the reaction catalyzed by OGG1 in the presence of 8-bromoguanine. A, nicked product formationfrom the AP substrate (1 �M) by C253I (squares) and C253L (triangles) OGG1 (1 �M) without (black symbols) or with 0.5 mM BrG (white symbols). B, nicked productformation from the 8-oxo-G substrate (1 �M) by C253I (squares) and C253L (triangles) OGG1 (1 �M) without BrG (black symbols), with 0.5 mM BrG added 30 s afterthe substrate (white symbols), and with 0.5 mM BrG preincubated with the enzyme for 45 min (gray symbols). C, nicked product formation from the AP substrate(1 �M) by wild-type (circles), C253I (squares), and C253L (triangles) OGG1 (0.2 �M) without (black symbols) or with 0.5 mM BrG (white symbols). Each symbol is themean of three independent experiments; S.E. did not exceed 20%; error bars are omitted for clarity.

TABLE 2Rate constant estimates for processing of oxoG and AP substrates by C253I and C253L OGG1

8-oxo-G APC253I C353L Wild typea C253I C253L

kglyc, s�1 (0.9 0.3) 10�3 (1.4 0.4) 10�3 0.08 NAb NA

kly, s�1 (4.0 1.0) 10�5 (1.8 0.5) 10�5 (0.7–1.4) 10�3 (11 3) 10�5 (8 2) 10�5

a Data are from Ref. 22.b NAmeans not applicable.



mechanical/molecular modeling results (14). Replacement ofcharged Lys-249 to the neutral one abandons the Lys-249

�

–Cys-253� dipole suggested to exist in wild-type OGG1 (14), but thesetwo residues are still held together by a strong hydrogen bond(�2.35 kcal/mol, 2.9 Å), existing over 99% of the simulation.All threemodels evolved slowly for the initial stage of the run,

up to 5–7 ns, and were quite stable after 7.5 ns, as evidenced byone- and two-dimensional r.m.s.d. plots (Fig. 5, B–E); overall,the heavy atom r.m.s.d. did not exceed 1.8 Å. Introduction of abulky substituent into the lesion recognition pocket, asexpected, displaced the 8-oxo-G base from the proper positionin the pocket. In the wild-type OGG1, Phe-319 forms one wallof the pocket, stacking with the everted 8-oxo-G. Notably, inOGG1 C253I, �80% of the area of the Phe-319 side chain over-laps with 8-oxo-G planar rings through the complete trajectoryof the dynamics, whereas in the C253L mutant, this overlap is�20% through the whole dynamics (Fig. 6A). Wild-type OGG1demonstrated an intermediate behavior, starting at�60%over-lap and then gradually decreasing to �20% at 4–6 ns. More-over, the inclination of the Phe-319 ring relative to the plane of8-oxo-G is �20° more in C253L OGG1 than in the other twomodels. The extra bulk of the isoleucine and leucine side chainis accommodated in different ways. In the C253I mutant, theside chain of Ile is folded to be generally parallel to the 8-oxo-Gplane, which may additionally restrain the base movementthrough van der Waals interactions, whereas the side chain of

Leu in C253L is extended and pushes the base further into thewall formed by Phe-319 (Fig. 6F).Rather unexpectedly, the Watson-Crick edge bonds with

Gln-315 were conserved in the mutants; the distances N1(oxo-dG0)–O�1(Gln-315) and N2(oxo-dG0)–O�1(Gln-315) werestable at �3.0 Å (with rare short breakages of the former bondin the mutants). The same was true for the N7(oxo-dG0)–O(Gly-42) hydrogen bond (�3.1 Å and �99% occurrence in allthree models).Close examination of the structural parameters of DNA

revealed that the mutations hardly affect the conformation ofthe oxo-dG nucleotide and the immediately adjacent DNA res-idues. Covalent bond torsion angles and deoxyribose phaseangle in these residues were almost identical in all three simu-lations, with minor changes in the � angle in C253L OGG1(approximately �20° turn in the second half of the simulation)and angle in C253I OGG1 (stays stable at �270° through thesimulation,whereaswild-type andC253Lmodels turn�20° in thesecond half of the simulation). Thus, paradoxically, it seems thatthe mutations in OGG1 that were supposed to occlude the base-binding pocket and prevent 8-oxo-G from entering it do not pre-vent full movement of the damaged nucleotide but rather distortthe pocket itself to accommodate the everted 8-oxo-G.The distance N(Lys-249)–C1�(oxo-dG0) is stable and simi-

lar (�4 Å) in all three models. However, the angle N(Lys-249)–C1�(oxo-dG0)–N9(oxo-dG0) is stably different between the wild

FIGURE 5. A, sequence of DNA duplex (PDB structure 1EBM) used in the modeling. Deoxynucleotides in the damaged strand are numbered starting from thecentral oxo-dG, the positive indices toward the 5� terminus and the negative ones toward the 3� terminus. In the complementary strand, deoxynucleotides arenumbered in the same manner starting from the central dC opposite the lesion; the indices corresponding to the complementary strand are in parentheses. B–E,general characteristics of molecular dynamic simulations of wild-type, C253I, and C253L OGG1. B, r.m.s.d. of wild-type (purple), C253I (magenta), and C253L(blue) OGG1. C–E, two-dimensional r.m.s.d. of wild-type (C), C253I (D), and C253L (E) OGG1.



type (�75°) and both mutants (�110°) (Fig. 6B). This angle isimportant for proper overlap of the orbitals during the nucleo-philic displacement of 8-oxo-G by the amino group of Lys-249,

and generally, such reactions tolerate no more than 10–15° dis-placement from the optimal alignment (33). However, the align-ment in the direction N(Lys-249)–C1�(oxo-dG0)–O4�(oxo-

FIGURE 6. A, overlap of Phe-319 and 8-oxo-G aromatic rings during the molecular dynamics in wild-type (purple), C253I (magenta), and C253L OGG1 (blue). Thetraces show moving average over 50 snapshots. B, angle N(Lys-249)–C1�(oxo-dG(0))–N9(oxo-dG(0)) of approach of catalytic Lys-249 of wild-type (purple), C253I(magenta), and C253L (blue) OGG1 to the reacting C1� of oxo-dG. C–E, population of the catalytically favorable conformation in wild-type OGG1 (C), OGG1 C253I(D), and OGG1 C253L (E). In the catalytically favorable conformation (top right cluster on each plot, very scarcely populated in E), Asp-268 makes a hydrogenbond with O4� of the damaged nucleotide and is far from O1P of the adjacent dGMP, whereas Lys-249 approaches C1� of the damaged nucleotide at a favorableangle for nucleophilic attack in the C1�–N9 direction. F, superposition of a representative simulation snapshot (5.9 ns) showing the overlap of the residuessandwiching 8-oxo-G in wild-type, C253I, and C253L structures. Note the position of Leu, which pushes 8-oxo-G and Phe-319 aside from their positions inwild-type and C253I OGG1. G, superposition of a representative simulation snapshot (7.0 ns) showing the distortion at the complementary DNA strandintroduced by active site-occluding mutations. Three central base pairs are shown. Tyr-203 wedge lies behind dC(�1). Note that dC(0) and dT(�1) are moredisordered than other overlapping bases. F and G, carbon atoms are colored according to the model: wild-type (purple), C253I (magenta), and C253L (blue).Oxygen atoms are colored red; nitrogen, dark blue; phosphorus, orange; and sulfur, yellow. Tyr-203 is completely colored according to the model.



dG0) (data not shown) is similar in all three models, consistentwith the idea thatN-glycosidic bond, and not C1�–O4� bond, isbroken first. The side chain of the second catalytic amino acid,Asp-268, in the wild-type enzyme remained near O4� 67% ofthe simulation and formed an alternative bondwith a nonbridg-ing oxygen O1P of dG�1 for 7% of the simulation. In themutants, however, Asp-268 was predominantly contactingthe O1P(dG�1) rather than O4�(oxo-dG0) (45 and 39% of thetime, respectively, for C253I OGG1, 60 and 27% of the time,respectively, for C253L OGG1; see Fig. 5A for the numerationof deoxynucleotides in theDNApart of themodel). Plotting thewhole trajectory in three dimensions with the coordinatesN(Lys-249)–C1�(oxo-dG0)–N9(oxo-dG0) angle, O�2(Asp-268)–O4�(oxo-dG0) distance, and O�2(Asp-268)–O1P(dG�1)distance, one can see that the active site populates four clustersof conformational space (Fig. 6, C–E), of which only one (topright) is favorable for catalysis with respect to Lys-249 attackangle and Asp-268 stabilization. This cluster is the most popu-lated in thewild-type enzyme (Fig. 6C), less visited by theC253Iactive site (Fig. 6D), and is almost empty in C253L (Fig. 6E).Thus, the combination of misalignment of Lys-249 and adecrease in the population of the catalytically competent Asp-268 conformation together may account for the compromisedcatalytic activity of the OGG1 mutants.Contacts with the strand opposite to the lesion are also

important for forming the properMichaelis complex byOGG1,reflected in the strong specificity of this enzyme for cytosine atthis position (34, 35). Two structural features of OGG1-DNAinteractions are the most eminent in fixing the proper confor-mation of the opposite strand. First, Tyr-203 side-chain wedgesbetweenCopposite to 8-oxo-G (dC(0)) and the base 5� to it (C in1EBM, dC(�1)), which helps to kink DNA. Second, the residuesAsn-149, Arg-154, and Arg-204 make contacts with dC(0).Analysis of dynamics of Tyr-203 inclination and overlap withthe interacting cytosines shows that all three structures do notundergomajor changes in this region. The distortion ofDNA inthe mutants is highest at the dC(0) and especially dT(�1) nucle-otides (Fig. 6G). However, this seems to affect only the back-bone, because all 6-bp parameters involving dT(�1) (shear,stretch, stagger, buckle, propeller, and opening) are overall notsignificantly different between wild-type and mutant OGG1.Regarding contacts with dC(0), the guanidine group of Arg-154in the C253I mutant drifts away from the base early in the sim-ulations, whereas in the C253L OGG1, Asn-149 is far from thecytosine in the first 80% of the dynamics but then assumes thewild-type-like conformation (data not shown).

DISCUSSION

A long-standing problem in the field of DNA repair is howDNA repairs enzymes, DNA glycosylases in particular, and dis-criminates their target-damaged nucleotides from the normalones, which are present in a great excess yet, with very rareexceptions, are all but refractory to the enzymes’ action. DNAglycosylases, as many other nucleic acid-dependent enzymes,enforce significant conformational changes upon their sub-strates. In the precatalytic complex, the helical axis of DNA iskinked at the point of the lesion (�70° in OGG1-DNA complex1EBM), and the damaged nucleotide is rotated from its position

inside the double helix into the enzyme’s active site. An emerg-ing model of glycosylase specificity acknowledges that nucleo-tides in DNA are sampled in several sequential steps, with nor-mal nucleotides rejected early in the process, whereas cognate-damaged nucleotides successfully clear all checkpoints andadopt a specific conformation and position in the active siteallowing theN-glycosidic bond breakage to ensue (25, 36). Thecheckpoints are transition energy barriers between the con-formers of the enzyme-substrate complex andmay correspondto DNA kinking, eversion of the damaged nucleotide, insertionof the enzyme’s amino acids in the void left after the eversion,and accommodation of the damagednucleotide in the enzyme’sactive site, etc.The substrate recognition and catalysis trajectory ofOGG1 is

well sampled by x-ray crystallography and molecular dynamicsimulation. In particular, four sequential conformations of thenucleotide eversion are known as follows: the early structurewhere the target base is disengaged from its Watson-Crickinteractions, unstacked, and slightly pushed toward the majorgroove (15); the intermediate structure where the target basefalls in a so-called “exo-site” at the entrance to the active site(14); the advanced structure where the target base is almost atits final position but has not yet formed all specific bonds withthe base-binding pocket of OGG1 (16, 17, 19); and the precata-lytic structure with the damaged base poised for removal (8, 11,16). Steered molecular dynamics with umbrella sampling indi-cates that all intermediate conformations are energetically sim-ilar with 2–4 kcal/mol barriers between them,whereas the finalfully everted conformation is 10–12 kcal/mol lower than theintermediates.5 For E. coli Fpg, another 8-oxoguanine-DNAglycosylase is structurally unrelated to OGG1, and the eversiontransition barrier directly determined from stopped-flow datahad enthalpy H0 � �15.5 kcal/mol, although that was par-tially offset by negative entropy attributed to the enzyme-struc-ture complex becomingmore rigid upon base eversion and voidfilling (25).In the precatalytic structure (8), the 8-oxo-G base stacks

against the aromatic system of Phe-319 and is pressed by thethiolate Cys-253� from the other side of the plane. The Wat-son-Crick-forming groups are all engaged in the interactionswith the enzyme’s active site; N2 donates one hydrogen bond toO�1 of Gln-315 and another to either O�1 of Asp-268 or themain chain O of Pro-266; N1 donates a hydrogen bond to O�1of Gln-315, and O6 accepts two hydrogen bonds from struc-tured water molecules that interact with other amino acid res-idues of the active site. Outside of the base-binding pocket butstill in the active site, Lys-249 is poised for the nucleophilicattack at C1�, and Asp-268 forms a hydrogen bond with O4� ofthe target nucleotide, assisting the deoxyribose ring opening andstabilizing the positive charge that develops on the ring when thebase leaves. The only bond specific for 8-oxo-G versusG is formedbyN7, which, in the pyrrolic type as in 8-oxo-G, donates a hydro-gen bond to the main chain O of Gly-42 but cannot do so in thepyridinic type as inG. Interestingly, ifG is forcibly presented to theactive site innearly the sameconformationas8-oxo-G, it is still not

5 C. Simmerling, personal communication.



excised (19). As the N-glycosidic bond in oxo-dG is chemicallymore stable than indG(37), this canonlybeexplainedby small butmultiple perturbations in the active site structurewhen the base inthe pocket is normal.Our interest in the mechanisms of substrate discrimination

by DNA glycosylases led us to address the question how a dis-tortion of the base-binding pocket can affect the action ofOGG1. We have chosen to replace Cys-253 with Leu or Ile,which are bulkier than Cys and should presumably displace8-oxo-G from its proper position within the active site. In addi-tion, the mutation should eliminate the negative charge on thethiolate anion, predicted from quantum mechanical modelingof the OGG1-DNA complex (14). The partial occlusion of theactive site of OGG1 by a Q315F mutation leads to a severelyreduced activity (16). Of note, OGG1-Q315F cross-linked toDNA at two different places produced two structures as fol-lows: one with 8-oxo-G in the exo-site and another with8-oxo-G in an advanced intermediate state; however, theamount of the residual activity was almost the same in bothcases (16). Our unpublished data6 on the Q315W mutant ofOGG1 confirm that collision of the active site with theWatson-Crick edge interferes with base excision. A recent report onanother active site-occluding mutant shows that the replace-ment of Cys-253 with Trp inactivates the glycosylase functionof OGG1 but spares the AP lyase activity (18).The minimal general kinetic scheme of OGG1 Cys-253

mutants turned out to be the same as the scheme for thewild-type OGG1 (22). Moreover, the profiles of fluorescenceincrease and decrease were consistent in the three forms of theenzyme, suggesting that the kinetic steps correspond to thesame conformational transitions. The rate constants for indi-vidual conformational transitions varied between the respec-tive constants for the wild-type enzyme; the differences wererather small at the early stages of the reaction, but the irrevers-ible step, ascribed to the catalytic process of N-glycosidic bondbreakage, was severely affected in themutants.We have observeda similar behavior of OGG1 and Fpg in our studies on substrateperturbation, in which good substrates (such as 8-oxo-G:C pairs)and poor substrates (such as 8-oxo-G:A pairs) were processed bythese glycosylases following the same kinetic scheme but with therates of one step being very different between good and poor sub-strates (24, 38). However, in the case of the poor 8-oxo-G:A sub-strate, the affected step was attributed to the insertion of OGG1residues into thevoid formedbydamagedbaseeversion (24); theseresidues formhighly specific discriminating bondswithC (8). Thelarge effect of pocket-occluding mutations on the N-glycosidicbond breakage clearly reflects the requirement for precise posi-tioning of catalytic residues relative to the reacting atoms of the8-oxo-G deoxynucleoside.A quite unexpected finding was stimulation of the activity of

the occlusion mutants by BrG. When this phenomenon wasfirst described for the wild-typeOGG1 and its AP lyase activity,it was assumed that the 7H-tautomer, assisted by the electro-negative bromine substituent, binds in the enzyme’s active sitein the samemode as the excised 8-oxo-G, and it acts as a general

base to pull a hydrogen off C2� to induce �-elimination (12).Later, however, it was shown that BrGalso efficiently stimulatesthe AP lyase activity of OGG1 when the substrate contains8-oxo-G, suggesting that the excised 8-oxo-G does not staytightly bound in the pocket but is in fast exchange with BrG(22). From the data we present here, it is apparent that theinitiation of �-elimination by BrG has relaxed geometricrequirements, because it is as efficient with the occluded base-binding pocket (5.5- and 15.2-fold stimulation for C253I andC253L, respectively) as with the wild-type one (9.2-fold stimu-lation). This is consistent with the observation that BrG-in-duced �-elimination has mixed stereochemistry of protonabstraction from C2�, with an �4:1 ratio of pro-R to pro-Shydrogens removed, indicating some flexibility of the active siteat this reaction step (12). Although we did not model OGG1-AP-DNA-BrG ternary complexes, the nearly identical confor-mations of the oxo-dG nucleoside in the active site of wild-typeandmutantOGG1 suggest that BrG also can be accommodatedin a similar way in all three structures.Another inference that can be made from the efficient stim-

ulation of the OGG1 C253L and C253I by BrG concerns thedepth of the 8-oxo-G sampling by the occlusion mutants.Indeed, it is highly unlikely that 8-oxo-G would be excised inthe exo-site, where the distances from C1� to the catalyticamino group of Lys-249 and carboxyl of Asp-268 are too long. Ifeven with the occluded (i.e. energetically unfavorable) activesite, �-elimination remains the rate-limiting step and can beenhanced by BrG, then 8-oxo-G populates its correct or nearlycorrect position in the active site of C253L and C253I with anon-negligible probability, much like in the case of the Q315Focclusion mutant with a distal disulfide cross-link (16) or thesubstrate containing photocaged 8-oxo-G (17). This inferenceis also supported by our modeling results, in which the catalyt-ically optimal spatial arrangement of Lys-249 and Asp-268 isless populated but still not completely prohibited in themutants (Fig. 6, C–E).Overall, both our experimental results and computational

simulations resonate well with the concepts of active site plas-ticity of enzymes (39, 40) and rugged free energy landscape ofenzymatic catalysis (41). Plasticity is characteristic of manyenzymes that have a range of substrates or catalyze differentreactions, thus having evolved to adopt a variety of conforma-tions and to stabilizemultiple, albeit similar, transition states. Ifthis is a case, mutation of a seemingly critical residue often isnot detrimental for at least some of the enzyme’s functions tothe extent that could be expected from the energy of the lostinteractions, because the active site is flexible enough to com-pensate partially for the misfit. Among DNA glycosylases,E. coliMutYs have been shown to compensate for a loss of Lys-20, a residue partially accounting for its weak �-eliminationactivity, by using another active site amine, that of Lys-142 (42).In our own work with E. coli endonuclease VIII,7 the mutationsabolishing critical interactions with DNA phosphates andgreatly diminishing the glycosylase activity have no effect onthe AP lyase activity due to restructuring of the active site that

6 M. V. Lukina, A. V. Popov, V. V. Koval, Y. N. Vorobjev, O. S. Fedorova, and D. O.Zharkov, unpublished observations.

7 G. Golan, D. O. Zharkov, A. P. Grollman, and G. Shoham, manuscript inpreparation.



fixes the substrate DNA in a catalytically competent conforma-tion. OGG1 has the ability to accommodate a moderatelydiverse set ofmodified bases, including 8-oxo-G, 8-oxoadenine,2,6-diamino-4-oxo-5-formamidopyrimidine, and several othersubstituted purines, and catalyzes base removal and �-elimina-tion, the latter capable of proceeding either with the excisedbase orwith no base in the active site (35, 43–46). Strikingly, theactive site of OGG1 is flexible enough to compensate for thepositional exchange of Lys-249 and Cys-253, resulting in anenzyme with a significant residual activity (18). We concludethat although base-binding pocket occlusion distorts the activesite OGG1 with the bound substrate and greatly decreases thecatalytic proficiency of the enzyme, it does not fully preventdamaged base sampling and excision, likely due to the intrinsicflexibility of the protein and the strong energetic preference for8-oxo-G eversion once the enzyme is bound to its preferredsubstrate DNA.

Acknowledgments—Computational time was provided by the Sibe-rian Supercomputing Center (NKS-30T cluster) as part of IntegrationProject 130 of the SiberianBranch of the RussianAcademy of Sciences.

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DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

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