The Mechanism of the Glycosylase Reaction with

The Supporting Information to

The Mechanism of the Glycosylase Reaction with hOGG1 Base-Excision Repair Enzyme: Concerted Effect of Lys249 and Asp268 During Excision of 8Oxoguanine Jakub Šebera1, Yoshikazu Hattori2, Daichi Sato3, David Řeha4, Radim Nencka1, Takashi Kohno5, Chojiro Kojima6,*, Yoshiyuki Tanaka2,*, and Vladimír Sychrovský1,7,*

1

The Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo 2 náměstí 2, 166 10, Praha, Czech Republic, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 3 Nishihama-Boji 180, Yamashiro-cho, Tokushima 770-8514, Japan, Graduate School of Pharmaceutical 4 Sciences, Tohoku University, Aobayama, Sendai 980-8578, Japan, Center for Nanobiology and Structural Biology, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Zámek 136, 373 33, Nové 5 Hrady, Czech Republic, Division of Genome Biology, National Cancer Center Research Institute 1-1, Tsukiji 56 chome, Chuo-ku, Tokyo, 104-0045, Japan, Graduate School of Engineering, Yokohama National University, 7 Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501, Japan, Department of Electrotechnology, Electrical Engineering Czech Technical University, Technická 2, 166 27, Praha, Czech Republic

The Supporting Information includes Supporting Theoretical Section Structural model, QM/MM calculation method, Calculation of Glycosylase reaction with hOGG1, Calculation of SN1 base-excision mechanism with hOGG1, Calculation of oxoG excision with hOGG1(D268N), Calculations of 2D potential energy surfaces, Calculations of atomic Fukui indices, Calculations of Riboseprotonated and N9-protonated mechanism employing original geometries from the Ref.(42), New calculation of the Ribose-protonated mechanism employing 2NOZ, The Ribose-protonated mechanism - summary Supporting Tables S1-S9 Supporting Figures S1-S6 Supporting Geometries: The QM/MM optimized geometries of QM parts for stationary states. Supporting References: Refs. 70-83

THE SUPPORTING THEORETICAL SECTION Structural model The x-ray structure with PDB ID 2NOZ (33) including hOGG1-DNA complex was used as initial structure for building the structural model used in QM/MM calculations. The DNA molecule was shortened and included seven base pairs with oxoG in the middle base pair. Only the x-ray resolved water molecules within catalytic core were kept (the four water molecules where the distance of O(H2O) atom from N9(oxoG) atom was smaller than 8 Å). The hydrogen atoms were added to protein and to DNA by employing the Leap (70) and Schrodinger Maestro (71), respectively. The solvated 2NOZ structural model was prepared by explicit solvation of 2NOZ. First, the x-ray unresolved loops Arg 79 — Ser 83 and Thr 285 — Ala 288 were complemented with YASARA (77). Than the hydrogen atoms were added to resulting structure employing Schrodinger Maestro. The complex was solvated with 25169 TIP3P (78) waters and Na+ ions within 93 Å x 93 Å x 93 Å cube box to maintain its electroneutral state. Geometry of bulk solvent only was equilibrated with molecular dynamics (MD) simulation employing GROMACS 4.5.5 (79-81): 100 ps NVT simulation followed by 100 ps NPT simulation with periodic boundary conditions. The amber99SB force field (82) was then used with exception of oxoG, which was parameterized by the RESP procedure (83) with HF/6-31G(d) atomic charges. The 2NOZ geometry of protein and A-oxoG-G tri-

nucleotide DNA fragment was constrained during all MD simulations using 10000 kJ mol-1 nm-2 force constant. The rest of DNA was constrained by 1000 kJ mol-1 nm-2 force constant and the solvent molecules with ions added to 2NOZ were without any constraint, allowing penetration of solvent into catalytic site. The solvated model used for QM/MM calculations included only the solvent molecules within the distance of 10 Å from oxoG, specifically 105 water molecules plus one x-ray resolved counter-ion Ca2+. Number of explicit water molecules within catalytic site increased upon complete hydration by two additional water molecules as compared to four already included x-ray resolved water molecules, which indicated nearly complete hydration of the catalytic site in 2NOZ. Total charge of the solvated structural model was 21 e-. Prior to QM/MM calculation of the R, TS1 and IS1 states one forward-backward scan along the C1’-N9 reaction coordinate was performed to achieve geometry/energy relaxation. The QM part in QM/MM calculations included Lys249(NH3+), Asp268(CO2-) and OxoG. QM/MM calculation method The QM and MM part of structural model was described employing Jaguar 8.2 (55),(56) and Impact 6.1 (57), respectively. The QSite 6.1 described QM-MM interface (58),(59). The structural model included hOGG1 protein, four water molecules within catalytic core and the DNA duplex including 7 base pairs. The geometries of all heavy atoms of undamaged DNA strand plus two terminal nucleosides of the damaged strand (214 atoms) were kept frozen. The central A-oxoG-G tri-nucleotide DNA fragment within damaged strand and whole hOGG1 protein were geometry optimized. The M06-2X (52) DFT functional and 6-31G(d,p) basis set (53) were used in calculations of QM part. The OPLS2005 all-atom force field was used in calculations of MM part (54). The QM part included oxoG nucleoside, Lys249 (53 atoms), and Asp268 (65 atoms), in some calculations also Gly42 (74 atoms). Validity of all stationary states was checked with the vibration frequency calculation. Electrostatic and steric effects were described by explicit protein-DNA environment. The Gibbs free energies included the harmonic vibration corrections (T=298.15 K and P=101325 Pa) calculated only for the QM part ended by hydrogen-atom caps, which was embedded within MM part. The QM calculations of Ribose-protonated and N9-protonated mechanisms described in the Ref. (42) employed the original QM geometries and were carried out with the Gaussian 09.B02 (72). New QM/MM calculations of Ribose-protonated mechanism employing 2NOZ structural model are described below. The atomic Fukui function F2 for N9 and C1’ atoms of oxoG were calculated with QSite (60-62). The negative value of F2 indicated nucleophilic character of atom whereas positive F2 indicated electrophilic character. Calculation of Glycosylase reaction with hOGG1 The QM/MM structural model included NH3+(Lys249) and CO2-(Asp268). The QM part included oxoG, Lys249 and Asp268. The x-ray 2NOZ geometry was geometry optimized. The C1’-N9 bond in oxoG was elongated with the step 0.1 Å and constrained during optimizations of grid point geometries until the N-glycosidic bond ruptured (C1’-N9 » 3.1 Å). The forward scan of C1’-N9 was followed by its backward scan up to reactant. The geometry was optimized without geometric constraint (C1’-N9 = 1.493 Å). The geometry-optimized reactant involving relaxed surrounding was then used for second forward scan calculation involving C1’-N9 distance. The relaxation of x-ray geometry owing to first forward scan was necessary for obtaining reliable QM/MM energy profile of oxoG excision. The relaxation affected particularly MM energy profile. Unprecedented decrease of the MM energies within the first forward scan resulted in spurious QM/MM energy profile with unreliably low energy barrier (Figure S2). Only the second forward scan resulted in MM energies that increased smoothly in response to departing oxoG base and the QM/MM energies described reliably the oxoG excision. Although the oxoG excision occurred within both forward scans roughly at C1’-N9 » 2.2 Å only the second forward scan resulted in unbiased QM/MM energy profile of the reaction. The geometry-relaxed and freely optimized reactant was used as reference state for calculation of oxoG base excision. The second forward scan involving C1’-N9 distance was performed and geometry calculated for the last point within the scan was freely geometry optimized (IS1 state, C1’-N9 = 2.993 Å). The R and IS1 stationary states and estimate of the interconnecting transition state calculated as TS1-maximum within scan (C1’-N9 = 2.2 Å) were employed in calculation of TS1 stationary state (C1’-N9 = 2.357 Å) by employing the Quadratic Synchronous Transit method. The R, TS1, and IS1 stationary states and all other stationary states along reaction pathway were validated with the calculations of harmonic vibration frequencies (Table S4). The interaction of lone pair electrons at N9(oxoG) atom with NH3+(Lys249) that can be foreseen already in 2NOZ (Ne-N9 = 3.03 Å) was key for the base-excision reaction. The proton of NH3+(Lys249) spontaneously transferred to N9 atom during the scan calculation involving C1’-N9 distance. The affinity of N9(oxoG) atom against proton of NH3+(Lys249) increased as the degree of N9-pyramidalization increased (κ‘ = 21.7° for R and κ‘ = 42.7° for TS1). After oxoG excision calculations continued towards formation of Schiff base (Lys249-ribose adduct with C1’-Ne double bond). The stationary state was first stabilized to prevent the backward reaction. The H9-Ne distance was stepwise elongated to obtain IS2 stationary state (H9-Ne = 1.906 Å (IS1) ® 4.290 Å (IS2)) that was calculated analogously as IS1: scan of the H9-Ne distance, optimization of IS2 and calculation of interconnecting TS2 state. The same procedure involving respective reaction coordinate was used in calculations of all remaining

stationary states. The IS2 described only rearrangement of cleaved oxoG base within catalytic site without departing from sugar (C1’-N9 = 2.681 Å). Afterwards calculations proceeded towards Lys249-ribose covalent adduct. The C1’-Ne distance was shortened and C1’- Ne bond was formed (2.910 Å (IS2) ® 1.487 Å (IS3)). The IS3 described Ne-protonated Lys249-ribose covalent adduct. The calculations continued towards the product of glycosylase reaction (the Schiff base). The reduced form of Schiff base captured in 1HU0 crystal described the state where oxoG base is cleaved and ribose of reduced Schiff base is opened (34). The proton of Ne-prononated Lys249-ribose covalent adduct was translated to oxoG base. The proton at Ne was first was abstracted by CO2-(Asp268) (IS4), which resulted in shortening of C1’- Ne bond (C1’- Ne = 1.487 Å (IS3) ® 1.441 Å (IS4)), and then translated to N3(oxoG) (IS5). The hydrogen bond between H9(oxoG) and O4’(ribose) facilitated opening of the ribose ring. The C1’-O4’ distance was elongated till the ring of ribose was opened (C1’-O4’ = 1.430 Å (IS5) ® 2.943 Å (IS6)) and protonated Schiff base was obtained (IS6). During the opening of ribose was H9(oxoG) proton abstracted by O4’(ribose) atom. The H9 proton of oxoG base was abstracted by O4’(ribose) during opening of sugar ring and the hydrogen at Ne spontaneously transferred to N9(oxoG) during optimization of product P. The geometry of product P complied with 1HU0 x-ray geometry (Figure 1d) except for the C1’- Ne distance that was formally double bond (C1’- Ne = 1.265 Å) whereas that in 1HU0 was single bond owing to borohydrate additive (C1’- Ne = 1.459 Å) (34). Furthermore, the calculated distance between O4’(ribose) and O atom of CO2-(Asp268) in P (3.586 Å) was longer than the O4’-O(Asp268) distance in 1HU0 (2.86 Å), which indicated that catalytic core for P need to be rearranged. Therefore, the O4’-O(Asp268) distance was stepwise shortened and resulting geometry optimized (P+1, (O4’-O(carboxylate) = 2.698 Å)). The geometric parameters (Table S2), thermodynamic parameters (Table S3), imaginary frequencies for TS stationary states (Table S4), Gibbs free activation and reaction energies (Table S5) and potential energy surfaces (Figures S2, S3) calculated for glycosylase reaction can be found in respective tables and figures. Calculation of SN1 base-excision mechanism with hOGG1 The 2NOZ structural model included NH2(Lys249) and CO2-(Asp268). The QM part included oxoG, Lys249 and Asp268. The initial geometry optimization of reactant was followed by forward and backward scan of C1’- N9 distance (oxoG) to obtain geometry-relaxed reactant (Table S9). The C1’- N9 distance was stepwise elongated up to C1’- N9 = 3.0 Å (Figure S3), however, stabilization of the intermediate state after excision of oxoG base was not calculated owing to lack of compensation of developing charge on leaving base and the SN1 mechanism therefore likely not occurs. Calculation of oxoG excision with hOGG1(D268N) The structural model included NH3+(Lys249) and Asn 268. Another structural model including + NH3 (Lys249) and CO2H(Asp268) was prepared and employed to illustrate effect of altered charge owing to protonation of Asp268 on oxoG excision. The mutation of original Asp268 to Asn was carried out keeping maximal overlap of the residues. The initial geometry optimization of reactant was followed by forward and backward scan of C1’- N9 distance to obtain geometry-relaxed reactant. The QM part included oxoG, Lys249 and residue 268 (Figure S3). The procedure that was used for calculation of σ-bond substitution reaction with WT hOGG1 was employed for calculations of IS1 and TS1 stationary states with D268N and CO2H(Asp268). Calculations of 2D potential energy surfaces The dependence of energy on C1’-N9 distance in oxoG and intermolecular distance between N9 (oxoG) and proton of NH3+(Lys249) was QM/MM calculated with the QM part including oxoG and NH3+(Lys249). The 2D potential energy surface was calculated for WT hOGG1 and hOGG1(D268N) mutant that were prepared and geometry optimized as described above. The 1D grid point geometries within second forward scan involving C1’-N9 distance in oxoG were used as starting geometries for calculations involving N9(oxoG)H(NH3+(Lys249)) distance that was varied within 1.5 Å ÷ 3.0 Å interval with the step 0.1 Å. The two geometric parameters were fixed for each geometry optimization within 2D potential surface. Calculations of atomic Fukui indices The Fukui indices calculated for N9 and C1’ atoms of oxoG (F2(N9), F2(C1’)) illustrated nucleophilic and electrophilic character of the atoms for F20, respectively. Nucleophilicity of the N9(oxoG) atom developed up to TS1 (C1’-N9 = 2.357 Å, Table S2) when proton of NH3+(Lys249) spontaneously transferred to N9(oxoG) (Figure S4, calculation for WT hOGG1). Increasing nucleophilicity of the N9(oxoG) atom with elongation of N-glycosidic bond illustrated increasing need for compensation of negative charge on departing oxoG base. Only the substitution of C1’-N9 bond with N9-H bond that occurred between the grid points C1’-N9 = 2.3 Å and C1’-N9 = 2.4 Å resulted in electro-neutral character of N9 atom of de facto nipped oxoG base. The initial nucleophilic character of N9(oxoG) atom was saturated during excision of oxoG base with proton that transferred from NH3+(Lys249). Interestingly, the C1’(ribose) atom preserved its electro-neutral character up to TS1 and became distinctively electrophilic only when C1’-N9 bond ruptured (Figure S5).

The dependences of F2(N9) and F2(C1’) on C1’-N9 distance calculated for hOGG1(D268N) mutant were similar as dependences for WT hOGG1 but the initial electronic characters of N9 and C1’ atoms changed long before TS1 (Figures S4 and S5). The N9 and C1’ atoms became both electrophilic between the grid points C1’-N9 = 1.9 Å and C1’-N9 = 2.0 Å. The same effect was calculated for CO2H(Asp268) (Figures S4 and S5). The proton transfer to N9(oxoG) from NH3+(Lys249) was hampered owing to both D268N and CO2H(Asp268) since the nucleophilic character of N9 atom was notably reduced. The loss of negative charge at the position of residue 268 hence results rather in SN1-like reaction despite favourable positioning of NH3+(Lys249) with respect to N9(oxoG). Moreover, the form of residue 268 affects electronic character of the N9 atom of oxoG base that accepts proton from NH3+(Lys249). The effects of Lys249 and Asp268 during oxoG excision are concerted in this way. Calculations of Ribose-protonated and N9-protonated mechanism employing original geometries from the Ref.(42) The QM geometries reported in the Ref.(42) were used in following calculations without any modification. The names of states in the Ref.(42) were used also in this work for describing N9-protonated mechanism: LRC1 (reactant) – Oxacarbenium maximum (base excision) – Oxacarbenium – SBI'-close maximum – SBI'-closed (product), and Ribose-protonated mechanism: LRC3 (reactant) – RP1 maximum (ribose-ring opening) - RP1 - RP2 maximum - RP2 - SBI maximum (base excision) – SBI (product). Note: (i) the structural model employed in the Ref.(42) was derived from 3IH7 x-ray structure that shows canonical G nucleoside enforcedly inserted into structurally corrupted hOGG1 catalytic site (29), (ii) the TS states in the Ref.(42) are not stationary states confirmed with vibration frequency calculations but rather energy maxima obtained within grid points on respective potential energy surfaces, (iii) the correspondence of N9-protonated mechanisms in the Ref.(42) with σ-bond substitution mechanism (46) anticipated by the authors in the Ref.(42) is incorrect (the explanation is given further). The original QM/MM (BLYP-D3/SVP:Amber) activation energy calculated for RP1 maximum within Ribose-protonated reaction was 18 kcal/mol (42). The activation energy for RP1 maximum calculated only for the QM part (employing QM geometries in the Ref.(42)) with BLYP-D3/SVP method was 29 kcal/mol. The RP1 activation energy calculated with M06-2X and B3LYP method was 46 kcal/mol and 35 kcal/mol, respectively (Table S7). The original QM/MM activation energy (RP1 maximum 18 kcal/mol) (42) was therefore probably calculated more likely owing to MM energies and not owing to QM energies that described chemistry within catalytic site. As for the N9-protonated mechanism (42), the distance of Ne(Lys249) atom from oxoG nucleoside in 3IH7 structure (Ne-N9 = 4.178 Å, Ne-N3 = 3.510 Å, Ne-C1’ = 4.293 Å) indicated rather the reactant of N3protonated mechanism(45) than reactant of σ-bond substitution mechanism(46). Use of the corrupted 3IH7 structure owing to enforcedly inserted incompatible G substrate most likely explains why the LCR1 state(42) (Ne-N9 = 3.211 Å, Ne-N3 = 2.687 Å, Ne-C1’ = 3.572 Å) failed to describe the key attack of lone-pair electrons at N9(oxoG) to NeH3+(Lys249). Moreover, the concept of σ-bond substitution reaction was likely misunderstood because the Fig. 4 in the Ref.(42) shows the C1‘ carbon of ribose attacked with NeH3+(Lys249) that would result during oxoG excision in repulsion between NeH3+(Lys249) and ribose oxocarbenium. The M06-2X method that is known for high-quality performance provided highest activation energies as compared to energies with other methods (Table S7). The activation energy for RP1 maximum within Riboseprotonated reaction and for Oxacarbenium maximum within N9-protonated mechanism was 46 kcal/mol and 40 kcal/mol, respectively. Interestingly, the RP1 maximum activation energy was larger than the Oxacarbenium maximum activation energy, which indicates easier excision with N9-protonated mechanism as compared to Ribose-protonated mechanism. New calculation of the Ribose-protonated mechanism employing 2NOZ The Ribose-protonated mechanism was QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculated employing 2NOZ structural model with the QM part including oxoG, NH2(Lys249) and CO2H(Asp268). The Ne-C1’ distance in oxoG was stepwise shortened after initial geometry optimization till the ring of sugar was opened (Ne-C1’ » 1.5 Å). Preliminary RP1 state was geometry optimized and than the backward scan of NeC1’ distance was calculated. The geometry for Ne-C1’ = 3.1 Å grid point within scan calculation was used for calculation of LCR3 stationary state. Second forward scan involving the Ne-C1’ distance employed the LCR3 geometry as initial geometry and resulted in optimization of the RP1 stationary state. The RP1 maximum within second scan and the LCR3 and RP1 stationary states were employed in the calculation (Quadratic Synchronous Transit method) of RP1 stationary state describing opening or ribose ring. Validity of all the stationary states was confirmed with vibration frequencies calculation (the Im frequency for RP1 can be found in Table S4). Comparison of the geometric parameters calculated newly in this work with original geometries from the Ref. (42) illustrated compatibility of the new calculations with original work particularly as regards the rate-limiting step of Ribose-protonated pathway (Table S6). The Gibbs free activation and reaction energy calculated for opening of ring of deoxyribose was 42.2 kcal/mol and 27.9 kcal/mol, respectively. The activation energy for RP1 stationary state was 43.3 kcal/mol.

The Ribose-protonated mechanism - summary The new calculations in Table S7 employing the original geometries for QM part indicated that the QM/MM energy profile in the Ref. (42) was obtained likely owing to MM energies and not as result of chemical changes within catalytic core calculated with QM energies. The M06-2X activation energy for RP1-maximum state calculated with original QM geometries was 46 kcal/mol (Table S7). The QM/MM (M06-2X/6-31G(d,p):OPLS2005) activation energy for RP1 stationary state calculated newly with the 2NOZ structural model was 43.3 kcal/mol. The activation energy calculated for QM part of original RP1-maximum state approximated the RP1 stationary state (calculated newly with 2NOZ) reliably. Hence, the QM/MM activation energy of RP1-maximum state 18 kcal/mol(42) was obtained most probably owing to MM energies and owing to BLYP-D3 that underestimated activation energies as compared to M06-2X or B3LYP activation energies (Table S7). The M06-2X provides reliable activation energies since this functional is well known for excellent performance for main-group thermochemistry and kinetic applications (52), and its application to glycosidic bond cleavage (73) and peptide bond formation (74) was also successful. The N9-protonated mechanism (42) is chemically incompatible with the σ-bond substitution mechanism (46). The N9-protonated mechanism corresponds likely to N3-protonated mechanism (45) that was compared with σ-bond substitution mechanism in the Ref.(46). Moreover, even the corrupted σ-bond substitution mechanism was superior to Ribose-protonated mechanism because when calculated with M06-2X or B3LYP method (Table S7). Plausibility of the true σ-bond substitution reaction therefore can’t be ruled out based on the calculations in the Ref.(42). Supporting Tables Table S1. The pKa values calculated for hOGG1 residues with the MOE 2014.09 program using the PROPKA method (75, 76). Residue

2NOZ1

R2

R3

Lys249

9.22

9.26

9.79

Asp268

3.09

4.64

4.45

1

2NOZ x-ray structure. QM/MM geometry optimized reactant with QM part including oxoG and Lys249. 3 QM/MM geometry optimized reactant with QM part including oxoG, Lys249 and Asp268. 2

Table S2. The QM/MM calculated geometric parameters for stationary states within the glycosylase reaction operated by hOGG1. State 1

C1’-N9

Nε-N9

Nε-C1’

Nε-N3

N9-H

Nε-H

H7-O(42) 2

C1’-O(268) 3

C1’-O4’

κ’

2NOZ

1.458

3.027

3.127

3.341

-

-

-

3.456

1.419

2.9

R

1.493

2.969

3.129

2.973

1.940

1.031

1.957

4.244

1.377

21.7

1.493

2.823

3.274

3.624

1.782

1.055

1.743

3.144

1.390

38.6

2.357

2.666

3.260

2.850

1.588

1.102

1.981

3.560

1.262

42.7

2.246

2.583

3.383

3.551

1.367

1.218

1.740

2.859

1.273

52.9

2.993

2.786

3.167

2.969

1.027

1.906

1.872

3.006

1.253

45.5

R

4

TS1 TS1

4

IS1 IS1

4

2.867

2.686

3.840

3.807

1.065

1.638

1.737

2.640

1.255

57.6

TS2

3.019

3.275

2.950

3.203

0.997

3.021

1.884

4.342

1.250

13.9

IS2

2.681

3.671

2.910

3.468

1.019

4.290

2.014

4.456

1.254

-47.4

TS3

3.216

3.160

2.215

3.024

1.015

3.392

1.962

5.141

1.283

-39.4

IS3

3.379

3.094

1.487

2.866

1.022

2.780

2.028

3.628

1.401

-24.2

TS4

3.351

3.053

1.492

2.928

1.022

2.736

2.036

3.614

1.402

-25.1

IS4

3.332

2.977

1.441

3.124

1.026

2.571

2.093

3.949

1.430

-33.7

TS5

3.215

2.864

1.438

3.115

1.036

2.487

2.052

4.054

1.440

-38.3

IS5

3.210

2.870

1.440

3.098

1.044

2.489

1.996

4.113

1.441

-39.3

TS6

3.143

2.855

1.297

3.469

1.884

2.882

2.099

4.341

2.192

-36.4

IS6

3.138

2.822

1.294

3.461

1.901

2.873

2.103

4.354

2.237

-34.8

TS7

3.114

2.726

1.286

3.420

1.962

2.895

2.149

4.351

2.364

-31.5

P

3.484

2.751

1.266

3.552

3.401

4.026

2.016

4.911

2.943

-26.0

P+1

3.489

2.701

1.265

3.423

4.177

4.460

1.965

4.278

3.188

-1.8

1HU0

3.716

2.702

1.459

3.596

-

-

-

4.605

2.845

1.4

1

The x-ray structures 2NOZ and 1HU0 and QM/MM calculated stationary states with the QM part including oxoG, NH3+(Lys249) and CO2-(Asp268). The inter-atomic distances in Å and the torsion angle κ’, κ’ = C4-N9-C1’-O8 -180°, in degrees. 2 The H7(oxoG)-O(Gly42) distance. 3 The C1’(ribose oxoG)-O(Asp268) distance. 4 Calculated for completely explicitly hydrated 2NOZ described in the section Structural model.

Table S3. The QM/MM calculated energies, enthalpies and Gibbs free energies in kcal/mol and entropies in cal/K.mol for stationary states within the glycosylase reaction operated by hOGG1 and the experimental thermodynamic parameters. State 1

∆E

∆H Related to preceding IS state

R

∆G

∆S

2

0

0

0

0

18.9

15.8

16.1

-0.9

20.0

16.6

15.9

2.1

IS1

9.1

7.3

6.3

3.5

IS1 6

8.2

6.7

5.5

4.1

TS2

9.7

7.8

6.7

3.6

IS2

2.4

1.5

0.6

3.

TS3

15.5

15.6

16.2

-2.1

IS3

-33.2

-30.9

-30.0

-3.3

TS4

1.6

1.0

2.4

-4.8

IS4

-7.8

-7.6

-7.4

-0.8

TS5

4.6

0.9

1.5

-2.3

IS5

1.0

-0.5

0.0

-1.5

TS6

16.7

17.0

17.7

-2.4

IS6

16.0

16.9

16.3

2.1

TS7

6.5

3.9

4.4

-1.4

P

2.3

1.5

0.7

2.4

P+1

-11.5

-10.2

-9.8

-1.3

TS1 TS1

6

Related to Reactant

3

P

-26.7

-27.8

-28.8

3.5

P+1

-38.7

-38.5

-39.5

3.5

Experiment Exp.

4

-

-

17.1

-

Exp.

5

-

18.6

19.6

-3.5

1

The QM/MM calculations with the QM part including oxoG, NH3+(Lys249) and CO2-(Asp268). 2 The thermodynamic parameters were referenced to the parameters for previous intermediate state. 3 The thermodynamic parameters were referenced to the parameters for reactant state. 4 Refs. (68),(69),(45) 5 Ref. (64) 6 Calculated for completely explicitly hydrated 2NOZ described in the section Structural model. Table S4. The QM/MM imaginary frequencies in cm-1 calculated for TS states within glycosylase reaction operated by hOGG1. TS State Imaginary Frequency

QM part

Base-excision mechanism

TS1

63.5

oxoG,NH3+(Lys249),CO2-(Asp268) σ-bond substitution

TS1

498.9

oxoG,NH3+(Lys249),CO2H (Asp268)

σ-bond substitution for protonated Asp268

TS1

26.5

oxoG, NH3+(Lys249), Asn268

σ-bond substitution for D268N

TS11

485.0

oxoG,NH3+(Lys249),CO2-(Asp268) σ-bond substitution

TS2

277.6

oxoG, NH3+(Lys249),CO2-(Asp268) -

TS3

275.8

oxoG, NH3+(Lys249),CO2-(Asp268) -

TS4

68.5

oxoG, NH3+(Lys249),CO2-(Asp268) -

TS5

924.2

oxoG, NH3+(Lys249),CO2-(Asp268) -

TS6

32.9

oxoG, NH3+(Lys249),CO2-(Asp268) -

TS7

193.3

oxoG, NH3+(Lys249),CO2-(Asp268) -

RP1

414.0

oxoG, NH2(Lys249),CO2H(Asp268) Ribose-protonated proposed in the Ref.(42)

1

Calculated for completely explicitly hydrated 2NOZ described in the section Structural model.

Table S5. The QM/MM Gibbs free activation and reaction energies calculated for excision of oxoG base via σbond substitution mechanism with WT and D268N hOGG1 in kcal/mol. hOGG1

Residue 268

∆G# 1

∆Gr 1

∆E 1

∆E 2

∆E 3

WT

CO2-(Asp)

16.1

6.3

18.1

18.1

19.5

WT

CO2H(Asp)

26.9

19.6

32.8

31.7

-

D268N

Asn

28.7

26.5

34.7

34.3

-

1

Calculated for the TS1 and IS1 stationary states with QM part including oxoG, NH 3+(Lys249) and residue 268. 2 Calculated as energy maximum within the scan calculation in the Figure S3 minus energy of reactant with QM part including oxoG, NH3+(Lys249) and CO2-(Asp268). 3 Calculated as energy maximum within scan calculation minus energy of reactant with QM part including oxoG, NH3+(Lys249), CO2-(Asp268), Gly42 and Gln43. Table S6. The interatomic distances in Angstroms calculated for oxoG excision with hOGG1 via Riboseprotonated mechanism proposed in the Ref.(42). State

C1’-N9

Nε-C1’

C1’-O4’

O4’-H(268)

O4’O(268)

O(268)H(268)

This work 1 LRC3

1.454

3.035

1.416

1.614

2.594

1.004

RP1 maximum

1.360

2.231

2.082

0.985

2.692

1.768

RP1

1.436

1.535

2.627

0.970

2.924

2.089

Geometric parameters from the Ref.(42) LRC3

1.454

3.109

1.464

1.529

2.552

1.030

RP1 maximum

1.370

2.252

2.223

1.029

2.546

1.521

RP1

1.441

1.564

2.773

0.999

2.613

1.615

1

The QM/MM calculated stationary states employing 2NOZ structure confirmed with vibration frequency calculation (Table S4). The QM part included oxoG, NH2(Lys249),CO2H(Asp268). The Asp268 was indicated with number “268”. Table S7. Comparison of relative electronic energies calculated with different methods in this work only for the QM part of original structural model in the Ref.(42) with original QM/MM relative electronic energies in the Ref.(42). State1

Referenc e State

BLYP-D3/ SVP2

M06-2X/ 631G(d,p)2

B3LYP/ 631G(d,p)2

BLYP/ 631G(d,p)2

BLYP-D3/ SVP:AMBER3

N9-protonated mechanism4 Oxacarbenium maximum (TS)

LRC1

33

40

31

25

35

SBI'-close maximum (TS)

Oxocarb enium

20

19

21

22

27

SBI'-closed (P)

LRC1

-12

-11

-14

-15

8

Ribose-protonated

4

RP1 maximum (TS)

LRC3

29

46

35

28

18

RP2 maximum (TS)

RP1

6

9

7

5

10

SBI maximum (TS)

RP2

-4

2

-4

-8

13

SBI (P)

LRC3

9

23

12

7

-1

1

The electronic energies calculated employing the original QM geometries from the Ref.(42) are in the Table S8. The relative energies in kcal/mol were calculated as energy of respective state minus energy of reference state. 2 The method and atomic basis used for QM calculations employing original geometry from the Ref.(42). 3 The original relative QM/MM energy from the Ref.(42). 4 The original definition from the Ref.(42). Note: the N9-protonated mechanism (42) and the σ-bond substitution mechanism are different base-excision mechanisms. Table S8. The electronic energies in a.u. calculated in this work with different methods employing the QM geometries from the Ref.(42). State1

Geometry2

BLYP-D3/SVP

M06-2X/ 6-31G(d,p)

B3LYP/ 6-31G(d,p)

BLYP/ 6-31G(d,p)

N9-protonated mechanism3 LRC1

Table SI.2

-1935.86158024

-1936.89854986

-1937.58720081

-1937.02685304

Oxacarbenium maximum

Table SI.3

-1935.80940132

-1936.83453222

-1937.53718102

-1936.98670294

Oxacarbenium

Table SI.4

-1935.82769963

-1936.84915740

-1937.55485838

-1937.00667928

SBI'-close maximum

Table SI.5

-1935.79566909

-1936.81963716

-1937.52196346

-1936.97231588

SBI'-closed

Table SI.6

-1935.88119346

-1936.91642035

-1937.60932872

-1937.05085887

Ribose-protonated mechanism

3

LCR3

Table SI.10

-1935.91747458

-1936.96511787

-1937.64565055

-1937.08114563

RP1 maximum

Table SI.11

-1935.87094171

-1936.89234647

-1937.59010448

-1937.03702036

RP1

Table SI.12

-1935.88686247

-1936.92387826

-1937.61303412

-1937.05212962

RP2 maximum

Table SI.13

-1935.87801777

-1936.90989189

-1937.60166180

-1937.04359682

RP2

Table SI.14

-1935.90099925

-1936.93750926

-1937.62734751

-1937.06453461

SBI maxi um

Table SI.15

-1935.90757155

-1936.93372764

-1937.63312510

-1937.07726959

SBI

Table SI.16

-1935.90287092

-1936.92835217

-1937.62633929

-1937.07046616

1

The original names of reaction states from the Ref.(42). Note: the N9-protonated mechanism(42) in fact does not correspond to σ-bond substitution mechanism. All calculations employing the Gaussian 09.B02 (72). 2 The number of table in the Supporting Information(42) including geometry of QM part. 3 The original definition from the Ref.(42). Note: the N9-protonated mechanism(42) and the σ-bond substitution mechanism are different base-excision mechanisms. Table S9. The QM/MM calculated geometric parameters for excision of oxoG base with hOGG1. State1

C1’-N9

Nε-N9

Nε-C1’

N9-H

Nε-H

C1’-O(268)2

κ'

4.454

6.3

SN1 mechanism (NH2(Lys249),CO2H(Asp268)) R

1.477

3.111

3.154

-

-

+

-

σ-bond mechanism (NH3 (Lys249),CO2 (Asp268)) R

1.493

2.969

3.129

1.940

1.031

4.244

21.7

TS1

2.357

2.666

3.260

1.588

1.102

3.560

42.7

IS1

2.993

2.786

3.167

1.027

1.906

3.006

45.5

+

σ-bond mechanism (NH3 (Lys249),CO2H(Asp268)) R

1.465

2.967

3.377

1.974

1.035

3.242

11.7

TS1

2.318

2.572

3.532

1.361

1.221

3.176

40.7

IS1

2.836

2.736

3.358

1.053

1.707

3.264

44.3

+

σ-bond mechanism (NH3 (Lys249), Asn268)

R

1.469

2.939

3.264

1.929

1.039

3.381

20.7

TS1

2.292

2.637

3.484

1.114

1.538

3.303

37.8

IS1

2.501

2.715

3.399

1.070

1.661

3.295

41.9

1

The QM/MM calculations with the QM part including oxoG, Lys249 and residue 268. All the interatomic distances in Angstroms and torsion angle κ’ = C4-N9-C1’-O8 -180° in degrees. 2 The distance between C1’ and N(NH2(Asn)) for D268N mutation.

Supporting Figures

Figure S1. The orientations of Asp268 with respect to oxoG nucleoside captured in different crystals. The distance between C1’(ribose) and O(Asp268) atoms was 3.456 Å (2NOZ), 4.199 Å (2NOF), 4.244 Å (QM/MM optimized Reactant), and 3.691 Å ÷ 4.153 Å for the 1EBM, 1YQR, 2NOB, 2NOE, 2NOH, 3KTU crystals indicated as X-ray group.

Figure S2. The dependence of QM/MM, QM, and MM energies relative to reactant on gradually elongated distance between C1’(ribose) and N9(oxoG) atoms calculated within first forward scan (left) and second forward scan (right).

Figure S3. The QM/MM calculated dependence of energy relative to reactant on C1’-N9 distance in oxoG. The QM part included oxoG, NH3+(Lys249),CO2-(Asp268) (σ-bond substitution reaction, red ●), oxoG, NH3+(Lys249), CO2-(Asp268) and Gly42 (σ-bond substitution reaction, red o), oxoG, NH3+(Lys249), CO2H(Asp268) (σ-bond substitution reaction for protonated Asp268, green ▲), oxoG, NH3+(Lys249), Asn268 (σ-bond substitution reaction for D268N mutation, blue▼), and oxoG, NH2(Lys249), CO2-(Asp268) (SN1 mechanism, black ■).

Figure S4. The QM/MM calculated dependence of Fukui index F2(N9) on C1’-N9 distance in oxoG. The QM part included oxoG, NH3+(Lys249),CO2-(Asp268) (σ-bond substitution reaction, red ●), oxoG, NH3+(Lys249), CO2H(Asp268) (σ-bond substitution reaction for protonated Asp268, green ▲), oxoG, NH3+(Lys249), Asn268 (σ-bond substitution reaction for D268N mutation, blue▼).

Figure S5. The QM/MM calculated dependence of Fukui index F2(C1’) on C1’-N9 distance in oxoG. The QM part included oxoG, NH3+(Lys249),CO2-(Asp268) (σ-bond substitution reaction, red ●), oxoG, NH3+(Lys249), CO2H(Asp268) (σ-bond substitution reaction for protonated Asp268, green ▲), oxoG, NH3+(Lys249), Asn268 (σ-bond substitution reaction for D268N mutation, blue▼).

Figure S6. The 1H-15N-HSQC spectra of WT hOGG1 and hOGG1(D268N) mutant measured on 600 MHz NMR at 298 K.

Supporting Geometries: QM/MM optimized geometries of the QM parts for stationary states. R – glycosylase reaction with WT hOGG1 O 25.28510100 23.13052300 36.50352900 C 24.13630200 23.14249400 37.33062200

C O C O C C N C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H

23.37145700 22.93689500 22.11245300 22.10399100 20.95503000 21.57084200 21.16080500 21.27551500 21.14538500 20.97903000 20.78341700 20.69217800 20.69190700 20.71888500 20.61676100 20.80044300 21.00783900 21.40040500 23.50578900 24.34422900 24.07270300 22.11422000 20.60897900 20.12355400 21.21931500 21.11575000 20.72370300 20.81767600 20.84151500 14.35971000 14.60107800 15.24975900 15.50947800 16.54411200 16.90303700 16.32373100 17.64358300 17.78020700 18.58545200 17.49174900 17.20185000 16.71236400 18.79755000 19.25664100 19.53751500 18.56033000 18.11041300 19.44039300 17.97112000 14.75125400 14.75721900 20.54898000 20.96725800

24.37194400 24.16532100 24.81890600 26.15891500 24.26439200 24.01728700 22.68654900 21.40749500 20.50720500 21.17257500 20.74013400 19.60868400 21.87014100 23.18782000 24.13992400 23.54349600 22.51072200 21.16401900 22.25928900 23.20340000 25.20887300 24.34081100 23.32013900 24.97390300 24.70260000 19.49916800 21.66366000 25.09693900 23.88547700 29.73096500 30.21151500 28.66446300 28.76905400 28.71737100 29.74865100 28.50228400 27.76192100 27.80733500 28.12272900 26.29069600 26.20193100 25.80867200 25.55823500 25.71918600 25.84747800 24.08561600 23.68299000 23.56434600 23.88187300 27.22946200 26.46483700 29.52864000 29.07044900

36.87772300 35.51601600 37.65416900 37.72820700 36.81675300 35.43131900 34.89394600 35.49466400 34.46803900 33.25002500 31.90751300 31.45883800 31.05412600 31.43326700 30.50019800 32.70424100 33.52394200 36.68422300 37.15475300 38.40008800 36.86362000 38.64187100 37.22644400 36.82303700 34.64996900 34.59780100 30.05768500 30.86352200 29.54412300 34.33574600 35.19715000 33.92236500 32.85511900 34.73145200 34.69426200 35.77742200 34.26890200 33.18708000 34.69590600 34.64999000 35.70524400 34.05943700 34.41113700 33.42409700 35.15648800 34.58183400 33.75618100 34.71203900 35.42414700 34.03073700 33.10069300 31.97224800 31.17738400

C H C H H C O O C O

21.35704100 20.87545400 21.51849000 22.32767200 20.59064500 21.71558900 22.69055800 20.78608600 22.62584000 22.81035900

29.46438000 30.00363700 27.98955100 27.78681500 27.68021400 27.19808600 27.55388900 26.40012400 30.25133300 31.05430200

33.17663800 34.01035100 33.62604800 34.34053900 34.11114500 32.31766600 31.63168800 32.00817600 32.98563000 32.11468800

TS1 – glycosylase reaction with WT hOGG1 O 25.15929500 23.20347400 36.40382800 C 24.02156200 23.22411500 37.23131000 C 23.42956600 24.60616600 37.01835100 O 23.00328700 24.66497300 35.58043200 C 22.15414500 25.05151100 37.74588900 O 22.14747800 26.38750100 37.82211900 C 21.06170500 24.54596000 36.80713200 C 21.74878000 24.59526800 35.46655200 N 20.80217900 22.51314300 34.89519000 C 21.00608600 21.26164300 35.45727000 N 21.05720400 20.33511100 34.43384500 C 20.88659200 20.99860300 33.21476300 C 20.75987700 20.59401200 31.86236300 O 20.74094400 19.47640200 31.36176300 N 20.64001800 21.74600200 31.03215900 C 20.58266100 23.04719400 31.44570800 N 20.55049700 24.03323000 30.52242900 N 20.52608900 23.36956100 32.71992600 C 20.75057000 22.32707900 33.54356200 O 21.10956300 21.02008800 36.66489100 H 23.29237200 22.46219200 36.91549300 H 24.21792500 23.09154200 38.29805100 H 24.20796700 25.36084100 37.09287000 H 22.09186000 24.54458800 38.71419200 H 20.84563200 23.49700300 37.02197000 H 20.14507300 25.13017000 36.88641700 H 21.36470500 24.78301000 34.45764400 H 21.08863300 19.32979400 34.57003800 H 20.72990500 21.56561300 30.03581800 H 20.87850300 24.92441600 30.92445800 H 20.87945600 23.78241900 29.59346100 N 14.36814700 29.69821900 34.33496900 H 14.61055200 30.18278600 35.19340900 C 15.25867300 28.63117200 33.92281600 H 15.52633000 28.73959000 32.85797000 C 16.54883800 28.66891100 34.74009400 H 16.92091900 29.69564800 34.70479600 H 16.32292000 28.45662400 35.78601000 C 17.63661000 27.69746100 34.27863000 H 17.76208300 27.74122400 33.19498700

H C H H C H H N H H H C O N H C H C H H C O O C O

18.58434400 17.48377200 17.16862300 16.72410100 18.80268700 19.26362500 19.52258000 18.62177600 18.27854700 19.54065600 17.94684900 14.75619800 14.76133600 20.56700300 21.01290100 21.39109000 20.91085800 21.58159500 22.33362800 20.63258800 21.90548900 22.75225100 21.21557900 22.65130800 22.81905500

28.05641600 26.22626000 26.14323900 25.73053200 25.49658500 25.61108900 25.87931500 24.03821100 23.58264700 23.47492200 23.85667100 27.19762600 26.43395400 29.46659500 29.02893600 29.47897300 30.04237600 28.02777300 27.88017700 27.69033700 27.17694300 27.67795000 26.13685000 30.26386300 31.05556100

34.69533800 34.66732500 35.71590400 34.06242000 34.48057800 33.49077700 35.20223000 34.73280400 33.88373500 34.96293600 35.50684000 34.02423200 33.09456100 32.00650800 31.21512700 33.19993400 34.01832900 33.70270500 34.49271600 34.12640900 32.45389800 31.68546100 32.30231000 32.96265700 32.07816900

IS1– glycosylase reaction with WT hOGG1 O 25.15119600 23.30662000 36.34843800 C 24.02251100 23.34700800 37.17317500 C 23.48678200 24.76662100 37.02639500 O 23.07509700 24.89707700 35.57633900 C 22.20449300 25.20299300 37.74263500 O 22.19778100 26.53814200 37.81985200 C 21.13563500 24.71338900 36.77058400 C 21.82971700 24.84338800 35.45355200 N 20.66125100 22.13818800 34.92950300 C 20.83593900 20.85110300 35.42382700 N 20.97162800 20.04772300 34.31961400 C 20.86405900 20.81925100 33.15331600 C 20.86589400 20.52218300 31.76336700 O 20.89770800 19.44081600 31.19413400 N 20.80973800 21.73485100 31.02286600 C 20.74277500 23.00258600 31.53463800 N 20.83589800 24.05781700 30.69853900 N 20.57127700 23.22989100 32.82178000 C 20.67694700 22.11630500 33.55683500 O 20.86771000 20.54997300 36.61026000 H 23.26373200 22.63106100 36.81887500 H 24.20528900 23.15992700 38.23413900 H 24.28455900 25.49356800 37.13658800 H 22.12531800 24.68969200 38.70560900 H 20.96169900 23.64550300 36.94138200 H 20.18296400 25.22916100 36.84738900

H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

21.43825200 21.03902600 20.98817900 21.21617700 21.19135300 14.32224700 14.56351400 15.21389900 15.46629600 16.51042600 16.86655600 16.29470200 17.58951800 17.65220900 18.55391800 17.48505200 17.18987500 16.73289800 18.82284600 19.22580900 19.54552500 18.76851100 18.51103500 20.10308600 18.02858500 14.73634600 14.75729900 20.67199900 21.11909400 21.51160200 21.03296400 21.73599700 22.41900800 20.77191500 22.19603500 22.85811600 21.80850500 22.75695500 22.90728300

24.90907000 19.03284300 21.64585900 24.88516300 23.86053600 29.71611000 30.19389500 28.64944500 28.74893900 28.72530000 29.75644400 28.53420300 27.74395600 27.71570400 28.13729000 26.30369700 26.28134700 25.73714300 25.59473900 25.67385400 26.12333500 24.18199000 23.65296400 22.87837900 24.01105300 27.21024400 26.43620100 29.38349900 28.93091500 29.44531400 30.01153100 28.01062100 27.91079600 27.63794900 27.11524700 27.69035700 25.91672700 30.24156300 31.02771100

34.42178900 34.36803100 30.02274400 31.18465000 29.76842100 34.33774200 35.20022400 33.92589700 32.85658300 34.72580500 34.65910400 35.77708200 34.27745600 33.18760400 34.62044800 34.76893100 35.82643300 34.21833000 34.61651200 33.59848600 35.25204200 34.97995600 34.15065400 35.37149300 35.67599700 34.04762100 33.12644300 32.07720100 31.29540100 33.25448700 34.07153400 33.78521300 34.64425300 34.13998600 32.61126100 31.72350600 32.64547900 32.98837200 32.09523800

TS2 – glycosylase reaction with WT hOGG1 O 25.09016900 23.19566600 36.35996300 C 23.96621600 23.24777100 37.18557200 C 23.44627700 24.67931500 37.04046900 O 23.05770300 24.82504800 35.57311100 C 22.16054700 25.14188100 37.73084700 O 22.15799400 26.47609900 37.81014400 C 21.07800000 24.73955400 36.72542300 C 21.81505400 24.84021600 35.43568300 N 21.29463100 21.92107200 34.86826400 C 21.33835900 20.63693800 35.38534400 N 21.17239000 19.82169500 34.29859600 C 21.06676200 20.59836600 33.13702900

C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

20.90754900 20.77047400 20.92183800 21.04891100 21.05363900 21.11943300 21.15377000 21.47514600 23.20525300 24.15300000 24.25559200 22.04326800 20.71827300 20.19279800 21.41961300 21.14484700 20.99786600 21.32003500 21.22439400 14.40817200 14.63114800 15.29700700 15.51284800 16.62635100 16.98246700 16.44349300 17.69744900 17.70482900 18.67678700 17.62740800 17.42508300 16.82516700 18.94974800 19.24093100 19.71870500 18.99613200 18.87257100 21.49864800 18.24859000 14.82565100 14.85554900 20.64629100 20.70485600 21.62416800 21.18728100 22.01637500 22.83618500 21.12875300 22.39793400 23.48135400 21.48734200 22.81058600 22.93928900

20.25531700 19.15702000 21.43579700 22.72752100 23.69912800 23.02085100 21.91905600 20.37758800 22.52241700 23.06825100 25.39241200 24.61877200 23.71429900 25.37163200 24.83589200 18.80381400 21.28481600 24.66484600 23.42841900 29.77927500 30.22893000 28.70767500 28.78901200 28.79601200 29.82615600 28.62546400 27.81342000 27.77006800 28.22044400 26.37271400 26.36506200 25.80500900 25.65931200 25.75464800 26.22292500 24.25309200 23.65991800 22.69747100 24.02775500 27.26502200 26.48406900 29.01762100 28.30652800 28.95207900 29.29242900 27.48262600 27.32988200 26.97717000 26.89259900 27.25839700 26.19366300 29.89301000 30.75675600

31.76802900 31.25510300 30.98831500 31.44117100 30.52441100 32.73595200 33.50365500 36.57248600 36.85345700 38.24707800 37.15873600 38.68484700 36.85554800 36.77152200 34.41318200 34.33184400 29.98138400 30.82374100 29.56514600 34.31481200 35.19828400 33.89847700 32.82187000 34.64798000 34.54614000 35.71050000 34.16676300 33.07490400 34.44500100 34.68422600 35.76402000 34.21234900 34.41279700 33.35849100 34.96881300 34.79257100 33.97898200 35.45969200 35.45934400 34.03195000 33.11536500 31.92669000 31.21210500 33.00532900 33.96409800 33.22385900 33.93934000 33.60319000 31.85809500 31.38911800 31.32257000 32.84131400 32.01796100

IS2 – glycosylase reaction with WT hOGG1 O 25.12543800 23.13996400 36.43619800 C 24.00520900 23.19427600 37.28220900 C 23.46277100 24.60452500 37.10147700 O 23.17018800 24.75359200 35.61879200 C 22.11643300 25.02424300 37.70705700 O 22.10818500 26.35923900 37.78494300 C 21.12738600 24.55769900 36.63208900 C 21.93790600 24.68509000 35.39641700 N 22.23692700 22.11554000 34.69124600 C 21.85356200 20.91385700 35.32562900 N 21.26889100 20.14603400 34.36031600 C 21.25609400 20.84484500 33.14646100 C 20.81505300 20.47460300 31.84731800 O 20.34579100 19.41465300 31.46272500 N 21.00234800 21.56048600 30.96999000 C 21.49681800 22.80865900 31.28768900 N 21.50121400 23.72336100 30.32363900 N 21.92892800 23.10464700 32.51255900 C 21.82707000 22.07202300 33.35774900 O 21.97905700 20.67823400 36.51728300 H 23.25908000 22.43784900 37.01399000 H 24.22527300 23.06147800 38.34245300 H 24.23591600 25.34432300 37.27874800 H 21.95843500 24.50340400 38.65725600 H 20.90508700 23.48666100 36.70778500 H 20.18444200 25.10026300 36.61948100 H 21.60676600 24.67221800 34.35528400 H 21.00707000 19.17062300 34.47660300 H 20.82752600 21.35509900 29.98990100 H 21.73471000 24.72271800 30.56583900 H 21.35853800 23.42271400 29.36964900 N 14.62313300 29.74749400 34.33461600 H 14.84794900 30.21830000 35.20654200 C 15.51413500 28.67613500 33.91582600 H 15.72979100 28.76619000 32.84050700 C 16.84849100 28.75612700 34.65750400 H 17.19178800 29.79346800 34.58894200 H 16.68564100 28.54179900 35.71689300 C 17.91735400 27.80814200 34.10547800 H 17.86555800 27.79350800 33.01684400 H 18.90251700 28.22239300 34.34814400 C 17.89977300 26.35338300 34.57692800 H 17.78393700 26.31903900 35.66878000 H 17.06693700 25.79133900 34.15515000 C 19.20416100 25.66138900 34.18167100 H 19.46900900 25.87274600 33.13741100 H 19.99801500 26.14870700 34.77303600 N 19.24329200 24.22017100 34.40183200 H 19.03324900 23.71652500 33.54734000 H 23.14192800 22.51174200 34.94026500 H 18.54818000 23.94537900 35.10377400

C O N H C H C H H C O O C O

15.05307900 15.08307500 20.80979800 20.84001800 21.79748700 21.36129200 22.26926000 23.13294700 21.42784800 22.59078300 23.70227500 21.60229900 22.94527700 23.06420000

27.23075000 26.45800500 28.85704900 28.12832700 28.78042200 29.05517500 27.31915700 27.17196400 26.76742300 26.75245400 26.99466400 26.16122400 29.76916100 30.64570000

34.03728400 33.11466200 31.92976000 31.22961500 32.99346600 33.97496200 33.13608400 33.79597400 33.54635900 31.74753900 31.27354500 31.20509000 32.86011300 32.04893800

TS3 – glycosylase reaction with WT hOGG1 O 25.07152800 23.22039600 36.45373900 C 23.95220600 23.26649200 37.30582800 C 23.41449500 24.67952800 37.14128400 O 22.98750200 24.90061200 35.70142800 C 22.18325900 25.19135500 37.90245600 O 22.18866600 26.52843600 37.98233700 C 21.05525400 24.88597900 36.92183700 C 21.74916200 25.23107500 35.64424500 N 22.13916100 22.18488700 34.69028000 C 21.83600500 20.95083600 35.28022200 N 21.36732700 20.16287600 34.26048400 C 21.38179800 20.88273500 33.06196900 C 20.98113900 20.54250500 31.73997500 O 20.55758100 19.48266900 31.30874700 N 21.14586000 21.66665700 30.90045300 C 21.54508900 22.92739400 31.27786800 N 21.50293000 23.89054600 30.35583600 N 21.90060300 23.21264400 32.52683400 C 21.84951900 22.14052200 33.33912600 O 21.92245300 20.68747000 36.46946500 H 23.21122600 22.50347400 37.03797900 H 24.18407100 23.12552300 38.36441600 H 24.23797500 25.38335200 37.23065300 H 22.08218200 24.66493900 38.85537800 H 20.82757100 23.81816800 36.86883500 H 20.15204200 25.46105400 37.11357500 H 21.55349900 25.96986300 34.88623900 H 21.15522300 19.17139900 34.34499600 H 21.02098000 21.48754000 29.90623000 H 21.91464900 24.83220600 30.57452000 H 21.47025000 23.60485800 29.38611600 N 14.81891200 29.68547600 34.34591700 H 15.04803400 30.17873900 35.20302300 C 15.71411600 28.61826300 33.92640600 H 15.95528100 28.72042700 32.85776100 C 17.02983900 28.69863900 34.70137000

H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

17.36510400 16.85162800 18.12324700 18.05461100 19.08645700 18.22268800 18.31189800 17.35308600 19.49015900 19.39735400 20.23968100 20.00117300 20.34007000 22.86700000 19.32000600 15.28166500 15.36328900 21.03695900 21.17779500 22.06436800 21.63378600 22.60534300 23.36100400 21.75992300 23.17869000 24.32768400 22.36349100 23.18833800 23.27583300

29.73970800 28.46785300 27.78625300 27.76104100 28.25920300 26.33670600 26.32581700 25.73141800 25.77629100 25.74800700 26.55820400 24.50348300 23.91566900 22.78249600 24.01058400 27.16579400 26.37432600 28.74955000 28.12840000 28.69520900 28.92009000 27.25076300 27.07925100 26.58930000 26.91346000 27.29898800 26.36273600 29.72424700 30.62312500

34.65384900 35.75518200 34.14797400 33.05722400 34.36486300 34.63907100 35.73276300 34.38081800 33.99867600 32.90938200 34.18636900 34.49386500 33.73573600 35.06762600 35.08154200 34.04388000 33.14060600 31.76639400 30.97877400 32.79499800 33.79210400 32.84476800 33.62092000 33.02198800 31.46537900 31.24112600 30.66677400 32.66973100 31.87915000

IS3 – glycosylase reaction with WT hOGG1 O 25.02183500 23.26832600 36.30391300 C 23.88411600 23.31787100 37.12842500 C 23.25313900 24.67896700 36.83971600 O 22.77726000 24.71358000 35.44056100 C 22.03301500 25.12856600 37.66592200 O 22.03589000 26.46846400 37.75258100 C 20.86493200 24.78747500 36.71577300 C 21.50719700 25.30458600 35.43893500 N 21.74430600 22.02835700 34.64830100 C 21.54294800 20.81248500 35.31089900 N 21.27019800 19.89537100 34.32275400 C 21.31101200 20.51998000 33.07092600 C 21.03989000 20.07377300 31.74720800 O 20.72415300 18.96815500 31.34244100 N 21.18601900 21.15244000 30.84119800 C 21.45969900 22.45381400 31.16042900 N 21.47531000 23.36553200 30.16945100 N 21.65554600 22.84100300 32.40537800 C 21.60391200 21.83922600 33.28962700 O 21.56684600 20.65230900 36.51921400 H 23.18291500 22.50309500 36.89145100 H 24.09674300 23.25651400 38.19841100

H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

24.03327200 21.98478100 20.70257300 19.94542200 21.57316100 21.11763900 21.11774100 21.82448500 21.67546700 14.99979600 15.22052400 15.89169700 16.08633400 17.24447400 17.53191300 17.12418700 18.32430300 17.97030100 19.21343400 18.80523500 19.32523300 17.97275700 19.74897200 19.17050300 20.27803600 20.82623200 21.51452500 22.32574800 20.48794200 15.51463400 15.73338400 21.22858900 21.50087800 22.25604300 21.79411800 22.86134700 23.94225400 22.41977900 22.62696000 23.06825600 21.97435300 23.31689400 23.39663700

25.43596600 24.60385800 23.70923000 25.28994400 26.39884900 18.90322100 20.89173000 24.28213500 23.02201100 29.64240600 30.13723300 28.56746600 28.64837800 28.67375200 29.72993100 28.37761900 27.82361700 27.53366900 28.43376000 26.54701100 26.82301700 25.89322800 25.87226700 25.28480800 26.63409000 24.94394200 24.97067700 22.77209700 23.98038700 27.10308500 26.23699800 28.89782700 28.46031900 28.89260300 29.11907000 27.46252400 27.42725500 26.97691200 26.61701900 27.07061900 25.54301400 29.96817900 30.83764600

36.93371500 38.62432000 36.62345100 37.00683800 35.45675800 34.47845200 29.86220500 30.47050600 29.23425900 34.37815000 35.23607500 33.95810200 32.87830200 34.67573000 34.67147600 35.72363500 33.99003500 32.99385700 33.84915600 34.69236300 35.61301400 34.96045800 33.69307900 32.97954400 33.11405600 34.16724800 33.32119400 35.03984400 34.23099200 34.15009400 33.34230700 31.75355700 30.88124000 32.78550800 33.76400800 32.94020100 33.11053000 33.80717000 31.68172600 30.61813700 31.84895100 32.67280200 31.84905200

TS4 – glycosylase reaction with WT hOGG1 O 25.01546300 23.33315300 36.29708400 C 23.87441600 23.38363000 37.11468400 C 23.23556400 24.73865900 36.81242000 O 22.73143600 24.76329500 35.42098400 C 22.03299200 25.18479700 37.66039600 O 22.03576900 26.52439000 37.74632600 C 20.84548600 24.82164200 36.73990500 C 21.44800000 25.32798300 35.44046400

N C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C

21.76834300 21.54954900 21.30104700 21.37532800 21.12562500 20.80070700 21.30280100 21.57849300 21.62021200 21.74848000 21.66744400 21.54269800 23.17825200 24.07908500 24.01519900 22.01096100 20.69274000 19.92869600 21.49162900 21.14436000 21.25118500 21.91401400 21.82748000 14.96398700 15.18300000 15.85690600 16.05448300 17.20614800 17.49973900 17.07923000 18.28420100 17.95032600 19.19139100 18.71400600 19.21568200 17.85808100 19.65805400 19.07174800 20.17998600 20.74860300 21.42453000 22.31093300 20.41828900 15.47559800 15.67218000 21.26336100 21.53042800 22.28191800 21.81727700 22.84196500 23.90060200 22.30468100 22.65715000

22.10003000 20.89210200 19.96694900 20.58013300 20.12540200 19.02039100 21.19371100 22.49690300 23.39216700 22.89261000 21.90065700 20.74230000 22.56406500 23.33396900 25.49888700 24.66277300 23.74022000 25.32027700 26.42350400 18.97666300 20.92884600 24.32791600 23.04107900 29.68167300 30.17557400 28.60827400 28.69235400 28.71379200 29.76823400 28.43186700 27.84774700 27.58681400 28.43921600 26.54659600 26.79015200 25.91099800 25.86625100 25.27465000 26.62595500 24.94722800 24.98209900 22.86138000 23.98184100 27.14494400 26.29055100 28.80655500 28.35207400 28.80249200 29.06768400 27.36707600 27.29839300 26.90486100 26.53577300

34.59917700 35.26635700 34.27974800 33.02380700 31.69884000 31.29804700 30.78758900 31.10196500 30.09792800 32.34789200 33.23830600 36.47735400 36.87914200 38.18690400 36.88398700 38.62134400 36.67164800 37.04565000 35.44965200 34.43957300 29.80827800 30.38925400 29.16767000 34.38979800 35.24966000 33.96988900 32.89129700 34.69182000 34.67452800 35.74189600 34.02552800 33.01455700 33.92107600 34.71400800 35.65339000 34.94941100 33.71560500 33.01200300 33.12585700 34.17928000 33.30636900 35.01290000 34.26112700 34.15137400 33.32510800 31.76416300 30.89952300 32.80714800 33.77472000 33.01396500 33.28676400 33.83450800 31.74523200

O O C O

23.21341900 21.88950200 23.35986700 23.44486200

26.94162100 25.53248300 29.85358900 30.71381900

30.71721400 31.85975200 32.67743500 31.84461800

IS4 – glycosylase reaction with WT hOGG1 O 25.02614600 23.36395100 36.28900300 C 23.88167300 23.41386100 37.10744300 C 23.23884300 24.77372800 36.81998200 O 22.67656700 24.79933800 35.46428000 C 22.08190500 25.22245600 37.72671600 O 22.08550200 26.56378700 37.81042700 C 20.85910400 24.83876500 36.88048800 C 21.34538500 25.31916100 35.52249200 N 21.80236300 22.18113700 34.49969900 C 21.51414700 21.01554500 35.20787800 N 21.20305400 20.07668000 34.24763700 C 21.25788800 20.65962800 32.98344100 C 20.93403600 20.16700400 31.69110600 O 20.55997400 19.06099500 31.34143300 N 21.10089300 21.19753600 30.73807200 C 21.47557200 22.48358300 30.99642100 N 21.52133000 23.33072500 29.93064300 N 21.73167600 22.92125100 32.20720300 C 21.62213300 21.97146500 33.15639300 O 21.49699400 20.88679300 36.42185600 H 23.18346600 22.60206500 36.85465100 H 24.09080200 23.34573500 38.17830900 H 24.02909100 25.52597100 36.86997600 H 22.11889400 24.71083700 38.69259900 H 20.71444600 23.75403100 36.83667500 H 19.94831400 25.33183600 37.21821400 H 21.37925300 26.42020600 35.52733100 H 21.00436100 19.09890900 34.43163400 H 20.98815200 20.90015800 29.77455700 H 22.02825800 24.18440900 30.13087700 H 21.73633800 22.88064900 29.04018100 N 14.91081400 29.67328100 34.37418900 H 15.13153800 30.16294000 35.23566200 C 15.80340600 28.59880700 33.95544200 H 16.00038400 28.67961100 32.87583500 C 17.15002500 28.71416500 34.67672200 H 17.43932300 29.76984500 34.64804300 H 17.02127300 28.44475400 35.72964000 C 18.23252100 27.83932100 34.03317800 H 17.93415100 27.59320300 33.00712800 H 19.15206400 28.41814700 33.97208600 C 18.62222400 26.53092300 34.72123000 H 19.07342900 26.75752700 35.69266600 H 17.75870900 25.88739900 34.90698500 C 19.64120500 25.83554500 33.79694900 H 19.08871500 25.27122300 33.04240900 H 20.17939500 26.62140900 33.24788900

N H H H C O N H C H C H H C O O C O

20.62146800 21.97550100 22.26029900 20.21635700 15.42098000 15.59469400 21.18822200 21.47178500 22.20878800 21.74882400 22.76259200 23.77536100 22.14138500 22.80106400 23.26980800 22.25840600 23.29941900 23.37709500

24.90960800 25.03709200 23.00761500 23.98808100 27.13619100 26.29174100 28.84222100 28.47090700 28.82164100 29.07075100 27.38067400 27.31443700 26.82837000 26.67422600 27.20320400 25.47507800 29.86582500 30.70980000

34.34516100 32.52365600 34.90027800 34.47657000 34.13625800 33.29544300 31.86227000 30.96590700 32.90256000 33.87484300 33.08811200 33.50375800 33.78537800 31.75834800 30.77236800 31.68388400 32.76090800 31.91197500

TS5 – glycosylase reaction with WT hOGG1 O 25.02881700 23.36704400 36.29279600 C 23.88524900 23.41637200 37.11394300 C 23.22636300 24.76740400 36.81575500 O 22.62849100 24.76158200 35.47499900 C 22.09088000 25.22363000 37.74591100 O 22.09472000 26.56439400 37.82814300 C 20.85035400 24.83164500 36.93783300 C 21.28599000 25.27609700 35.55476500 N 21.85858900 22.29141600 34.50675600 C 21.54993600 21.11407700 35.19258300 N 21.22254100 20.20271700 34.21276100 C 21.27757400 20.81582800 32.96827200 C 20.94924300 20.32327700 31.67953600 O 20.56377900 19.22406600 31.33291300 N 21.13258300 21.35234100 30.72342400 C 21.51448700 22.63117400 30.96127600 N 21.58747800 23.45944800 29.90538400 N 21.76435100 23.06795800 32.19596300 C 21.65511600 22.11959400 33.17323200 O 21.52902700 20.95698800 36.40137200 H 23.19358200 22.59636800 36.87303300 H 24.09844900 23.36059500 38.18471100 H 24.01342300 25.52448300 36.83232700 H 22.15195400 24.71457000 38.71165600 H 20.70335200 23.74637400 36.91390000 H 19.94701300 25.33185600 37.28490700 H 21.33286500 26.37389300 35.51525600 H 21.02629600 19.21786100 34.36708300 H 21.03865500 21.05441100 29.75660300 H 22.04207600 24.35339600 30.09228300 H 21.72884200 23.00611900 29.00408300 N 14.91274600 29.66391100 34.36787000 H 15.13525100 30.15544100 35.22757500

C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

15.80551300 15.99909400 17.15514000 17.44715900 17.02976300 18.23080600 17.93436700 19.15803200 18.59494000 19.04662200 17.71920100 19.60587300 19.05293800 20.20724500 20.49763700 22.06630800 22.27150700 19.98438800 15.42070200 15.58863300 21.18106800 21.45959000 22.19681700 21.74240300 22.73594700 23.74637400 22.11153500 22.78438600 23.21527900 22.32456800 23.29006300 23.37263700

28.58985100 28.66987500 28.70502100 29.75983100 28.43939300 27.82114700 27.57972600 28.38754500 26.51324800 26.74229900 25.88788100 25.77177100 25.22564700 26.51449900 24.81040400 24.31706400 23.13845000 23.97311700 27.12770200 26.28357200 28.78124300 28.38447100 28.71865200 28.96647300 27.26691200 27.21209500 26.71175100 26.54579700 27.15218500 25.34197300 29.75957200 30.60747800

33.94849500 32.86834800 34.66349600 34.63074000 35.71821600 34.01955900 32.99199900 33.96400600 34.71509100 35.68670300 34.90618900 33.82140800 33.05250600 33.28207600 34.44568900 32.24004500 34.93731800 34.70265600 34.12933600 33.28676900 31.83816100 30.95138400 32.87797100 33.85428200 33.04477600 33.46900700 33.73606100 31.70966500 30.73317100 31.63684500 32.74285000 31.89656600

IS5 – glycosylase reaction with WT hOGG1 O 25.02711100 23.35622600 36.29362800 C 23.88502500 23.40334500 37.11585600 C 23.20329000 24.73745500 36.79049300 O 22.56245000 24.66785600 35.47187400 C 22.09553900 25.21557300 37.74288600 O 22.10094900 26.55594600 37.82355200 C 20.82907600 24.82290300 36.97614200 C 21.23025200 25.20970100 35.56629100 N 21.83174900 22.22056100 34.56370300 C 21.52258800 21.02855700 35.22314800 N 21.20192100 20.13413800 34.22113100 C 21.26984200 20.76832600 32.98512000 C 20.96778800 20.32332700 31.66932900 O 20.58766600 19.23998800 31.27918500 N 21.17641900 21.38261500 30.73518100 C 21.53333700 22.65995200 30.99821200 N 21.64900500 23.55255700 30.02974400 N 21.73940300 23.02331500 32.27706100 C 21.63525200 22.06143000 33.23792300

O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

21.50048100 23.20325000 24.10106800 23.98383800 22.17856900 20.65826700 19.94723500 21.29956300 21.02710800 21.12437400 22.03230800 21.71893300 14.92674300 15.15114900 15.81869200 16.00154300 17.17788800 17.47684000 17.06294100 18.23701600 17.92562400 19.17411900 18.57795600 19.03860100 17.68748200 19.56175600 18.98948800 20.21151700 20.39649500 21.96175100 22.22136500 19.81234000 15.43453500 15.60041800 21.16348900 21.45210300 22.19325300 21.74419000 22.75419200 23.76931800 22.13740100 22.78263900 23.22665000 22.30545200 23.26901500 23.34707200

20.85453000 22.56843000 23.37055100 25.50063800 24.71207800 23.74142600 25.35354600 26.30316500 19.14168600 21.10645800 24.48630300 23.19421900 29.65130600 30.14360800 28.57694100 28.65303100 28.69458400 29.74659600 28.44286900 27.79176100 27.53849400 28.33879800 26.48935500 26.73017200 25.89181700 25.68718200 25.14281400 26.37688400 24.70486400 24.02785800 23.08579700 23.94918400 27.11520300 26.26773800 28.81278300 28.42667600 28.77587700 29.00306500 27.33219600 27.26093200 26.75239800 26.63275600 27.26126400 25.45316600 29.83410600 30.69929800

36.42747500 36.89370800 38.18683300 36.75087000 38.70993800 36.99222600 37.33332400 35.47807000 34.35656400 29.75721700 30.34095400 29.08193800 34.35202800 35.21040700 33.93075100 32.84827700 34.62742400 34.57741600 35.68718200 33.97966600 32.95947100 33.90635200 34.69835900 35.66339800 34.91039900 33.82870200 33.07172900 33.27622300 34.50573200 32.46831900 34.99892200 34.85566200 34.11807800 33.27826300 31.82639700 30.93662700 32.85324300 33.83747100 32.98175200 33.39277900 33.66340200 31.62563800 30.65911400 31.60370000 32.72103800 31.89211400

TS6 – glycosylase reaction with WT hOGG1 O 25.10155000 23.24005600 36.28210600 C 23.95629300 23.28028300 37.09962000 C 23.31811300 24.64059300 36.80165800 O 22.82720500 24.72544600 35.44715400 C 22.15650000 25.06310800 37.71559800

O C C N C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C

22.14939800 20.88099800 20.70763200 21.52231400 21.27282600 21.09454800 21.21552300 20.95158400 20.59706500 21.15073400 21.48081600 21.63397000 21.60874900 21.47238000 21.17621700 23.25725000 24.16031400 24.09994700 22.27741800 20.89324600 20.00694500 20.97634400 20.95598300 21.12454100 22.02070400 21.78576700 14.79843100 15.01796300 15.68522600 15.84201800 17.06105700 17.38119700 16.97173200 18.07712900 17.77655000 19.06114800 18.27059400 18.68731600 17.34153100 19.24480700 18.71414700 19.99458400 19.91216100 21.91932900 22.62897000 19.62176100 15.28288900 15.41776100 20.99320800 21.30958900 22.02747800 21.59077700 22.53562600

26.40457200 24.54249800 25.19128600 22.37290100 21.16586700 20.18677000 20.77908800 20.31766600 19.23086900 21.38324800 22.66067700 23.54180200 23.04839600 22.10651800 20.99597500 22.47117500 23.21280500 25.39467800 24.58052200 23.45216200 24.84371500 26.24166400 19.19601600 21.09349500 24.46050000 23.15678200 29.73761400 30.21140000 28.66495500 28.73236900 28.78252000 29.82452900 28.56161400 27.83362900 27.61121400 28.28819900 26.51159000 26.73628400 25.97115700 25.59115200 24.95021500 26.15515600 24.72108000 24.02788600 23.80658300 23.75088500 27.20602100 26.37384400 28.83612200 28.47698200 28.81606500 29.08602800 27.36318000

37.79170800 37.07003800 35.75851900 34.63133200 35.26613500 34.29525500 33.04429100 31.73638400 31.32031400 30.78941800 31.05064600 30.06375100 32.32682300 33.32489500 36.48340100 36.83291500 38.17198900 36.90416300 38.68996700 36.95312600 37.65667100 35.67242000 34.46145400 29.81565000 30.34946100 29.13473100 34.32335400 35.19477300 33.89782100 32.81039200 34.55189800 34.46273100 35.62043000 33.90288600 32.87582900 33.89074500 34.63672900 35.62456100 34.81483400 33.88726600 33.18284600 33.32060300 34.84877900 32.49193600 35.14937600 34.93566200 34.08377000 33.22285900 31.96456900 31.07201400 32.99340700 33.97231300 33.15132800

H H C O O C O

23.48980100 21.82446300 22.67567000 23.13278600 22.26653100 23.12940400 23.20986700

27.23410300 26.79859800 26.68896000 27.37068600 25.49007100 29.84266200 30.69951600

33.68020500 33.75262500 31.78623000 30.86009400 31.71788100 32.82411700 31.98700800

IS6 – glycosylase reaction with WT hOGG1 O 25.12425700 23.22748600 36.28720000 C 23.97746300 23.26570000 37.10231400 C 23.33075500 24.62341900 36.80751000 O 22.83784900 24.71848400 35.45654700 C 22.16687400 25.03767200 37.72515700 O 22.15826300 26.37916500 37.80274300 C 20.89261600 24.51186600 37.07854100 C 20.67950100 25.19913700 35.79312700 N 21.45505700 22.39197000 34.62494200 C 21.21480300 21.18508000 35.26260900 N 21.05549100 20.19957200 34.29573000 C 21.17602800 20.78858800 33.04309700 C 20.94686800 20.31340300 31.73305700 O 20.62957900 19.21544200 31.31755200 N 21.13669500 21.37795900 30.78334200 C 21.43447200 22.66348600 31.04267800 N 21.58778200 23.54295100 30.05372800 N 21.52963700 23.06205900 32.31838800 C 21.41134700 22.12028000 33.31827000 O 21.11070600 21.02150200 36.48034300 H 23.28157100 22.45405300 36.83188000 H 24.17968200 23.19549900 38.17507800 H 24.10912800 25.38053500 36.91428100 H 22.28746800 24.55281900 38.69866200 H 20.92811200 23.42540200 36.92992100 H 20.01852600 24.76950400 37.68553900 H 20.93135900 26.25603600 35.73456700 H 20.94668800 19.20593300 34.46640100 H 21.14201200 21.08065800 29.81115100 H 21.96849500 24.46256400 30.34381000 H 21.76697300 23.15437000 29.13078800 N 14.76880300 29.74635800 34.32894200 H 14.98863800 30.21973300 35.20074200 C 15.65497300 28.67297900 33.90390100 H 15.81368300 28.74127500 32.81656700 C 17.02985600 28.78486300 34.56068100 H 17.35680800 29.82468300 34.46888800 H 16.93642100 28.56792700 35.62933500 C 18.04299600 27.82858500 33.91740900 H 17.74605700 27.60642100 32.88929900 H 19.02925200 28.27910600 33.90789800 C 18.22876000 26.50416900 34.65098200 H 18.62591300 26.72410800 35.64787700 H 17.29871800 25.95921500 34.80796900

C H H N H H H C O N H C H C H H C O O C O

19.21841500 18.69656900 19.95701300 19.89859500 21.82437700 22.60394600 19.66639200 15.24811700 15.37840200 20.95826400 21.27525100 21.98985800 21.55212600 22.48980800 23.44351100 21.77539600 22.62697100 23.11039200 22.18649300 23.09743600 23.18073300

25.59250900 24.93768800 26.16270200 24.73297000 24.04868600 23.80865800 23.74371600 27.21539900 26.38559300 28.83136700 28.47385500 28.80790900 29.08306400 27.35285200 27.21948600 26.79375600 26.68358000 27.36066000 25.49638600 29.82840500 30.68405400

33.90981300 33.21156000 33.33448100 34.87241100 32.48106700 35.16073700 34.92025100 34.08807100 33.22415900 31.97802200 31.08506800 33.00984300 33.98664800 33.17155000 33.70000000 33.77414300 31.80411300 30.88841900 31.71936500 32.83915900 32.00111000

TS7 – glycosylase reaction with WT hOGG1 O 25.10892200 23.20934300 36.30414900 C 23.95877200 23.25093100 37.11428300 C 23.33665500 24.62364900 36.83576900 O 22.88787400 24.77289400 35.47778800 C 22.15927800 25.02830400 37.73906400 O 22.14943900 26.37072800 37.81571900 C 20.88978100 24.48678400 37.09548200 C 20.59680100 25.21488100 35.85532700 N 21.38795000 22.47527500 34.60323900 C 21.19392200 21.26343500 35.23903500 N 21.08922300 20.27193900 34.27631400 C 21.18662800 20.86786300 33.02706900 C 20.98375700 20.35989100 31.72668700 O 20.71317900 19.23783100 31.32953400 N 21.13464100 21.41528900 30.78515300 C 21.39284300 22.71715500 31.05632200 N 21.50625000 23.53771600 29.97762100 N 21.47619600 23.18235300 32.29447300 C 21.37019300 22.21793200 33.27747600 O 21.08738900 21.09129800 36.46024600 H 23.25431200 22.45346400 36.82557600 H 24.15396000 23.16157200 38.18720400 H 24.12144500 25.36601600 36.98914700 H 22.27810000 24.54584900 38.71415500 H 20.94903700 23.40608200 36.91502800 H 20.02174900 24.70037200 37.72982900 H 20.78256900 26.28778700 35.84891300 H 20.98333800 19.27822200 34.44514200 H 21.15023200 21.11688100 29.81440500 H 21.97636400 24.41615800 30.16073400

H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

21.75195400 14.77485000 14.99261000 15.66026900 15.82179700 17.03336900 17.36562100 16.93239500 18.04956300 17.77020900 19.03993400 18.20707000 18.58254900 17.27340100 19.20451100 18.69791600 19.93588500 19.88784100 21.82983400 22.64477500 19.85255200 15.24819500 15.37415200 20.97400700 21.27398100 21.99602600 21.55041600 22.48095700 23.44036100 21.77056100 22.60502800 23.19985800 21.94122400 23.11684800 23.19541400

23.06060900 29.74731300 30.21882100 28.67182300 28.73914900 28.77982800 29.81772000 28.56920800 27.81501200 27.60119200 28.25862000 26.48478200 26.69264600 25.94150500 25.58131600 24.92213800 26.16614200 24.73212700 24.61360600 23.88227700 23.70773300 27.21478400 26.38785400 28.80993000 28.44826800 28.78707900 29.08939400 27.32743700 27.21159900 26.79377000 26.64940500 27.22202800 25.54264900 29.79836700 30.63661100

29.11132000 34.33187200 35.20532500 33.90842500 32.82131800 34.57116200 34.47617400 35.64014500 33.94359400 32.90879400 33.95469600 34.66828800 35.67522100 34.80034200 33.93103600 33.22672300 33.36093300 34.89647300 32.29292000 35.14091800 34.83334000 34.09136900 33.22446200 32.01253900 31.11666900 33.05823700 34.02174400 33.28569000 33.80630700 33.91395900 31.93868400 31.04197300 31.73038400 32.87900600 32.02421300

P – glycosylase reaction with WT hOGG1 O 25.07351200 23.30585100 36.22781500 C 23.92686200 23.36468900 37.05705600 C 23.39753600 24.78231400 36.80338600 O 23.07385100 24.89914500 35.41053700 C 22.22047900 25.23306500 37.65436300 O 22.21356000 26.57499500 37.72768200 C 20.89322500 24.65697200 37.16789400 C 20.23926700 25.37533800 36.04476000 N 21.09559300 22.40124800 34.44457600 C 20.93096000 21.18311100 35.08688500 N 20.92449800 20.23424800 34.08638400 C 21.02539900 20.86508800 32.84831900 C 20.90380400 20.38670500 31.51815600 O 20.70797100 19.26213300 31.09488500 N 21.03708000 21.47772900 30.61776900 C 21.24361600 22.78194600 30.94432400

N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O

21.41439000 21.23900800 21.12008400 20.81484900 23.19146200 24.13596900 24.21518800 22.40126200 21.01416300 20.17445400 20.08736500 20.87451700 21.13636500 21.93835400 21.68799100 14.61056100 14.83760300 15.49306100 15.69259700 16.84086200 17.22090100 16.69382100 17.84797900 17.68461800 18.85797800 17.84972400 18.00378200 16.90733100 18.95450100 18.51518100 19.58309900 19.78373800 22.22535800 23.79438300 20.74284000 15.02329500 15.07770400 20.76061000 21.08010400 21.77838600 21.35368100 22.22768300 22.97305700 21.38211400 22.76518600 23.17409000 22.80730600 22.94241300 23.02569600

23.65006000 23.21424900 22.21154900 21.01636200 22.61847600 23.21114800 25.46988300 24.77595200 23.59874400 24.72654800 26.45492700 19.22929500 21.21219000 24.48100800 23.21578300 29.73457100 30.20887000 28.65775300 28.73700500 28.70868300 29.73141200 28.50853000 27.70110600 27.57982800 28.08861800 26.30472900 26.39470200 25.78227800 25.43654200 24.67016500 26.05724500 24.74268400 24.73724400 24.39647300 23.29772300 27.21133800 26.42094400 28.68449400 28.36558400 28.61903300 28.91061600 27.15703700 26.96953500 26.55081400 26.62852800 27.37706200 25.31984300 29.57274200 30.39576000

29.91009800 32.19016200 33.09691100 36.29406400 36.71963200 38.11780500 37.03922100 38.63550300 36.91811200 37.99017900 36.17444300 34.22645400 29.63978100 30.15830100 29.03095300 34.35625200 35.22588100 33.93854100 32.85786600 34.65361800 34.56872200 35.71850200 34.08083300 33.00802300 34.21998700 34.70158600 35.78016500 34.57380200 34.07260300 33.43013400 33.41823300 35.04753900 32.41579400 34.99356700 34.83651700 34.08130000 33.17477300 32.14680300 31.24199400 33.18883900 34.16226500 33.36083400 34.14987700 33.68913800 32.05335600 31.19029100 31.85930300 32.96880400 32.09748200

P+1 – glycosylase reaction with WT hOGG1 O 25.03233200 23.24621400 36.22826600 C 23.88331600 23.29544800 37.05722300

C O C O C C N C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H

23.37436600 23.15721600 22.17759900 22.16977000 20.84281800 20.08435000 20.46234600 20.51614600 20.63141800 20.64679200 20.72462600 20.73367500 20.80704900 20.81015400 21.03251700 20.58677200 20.54429800 20.47564500 23.14697700 24.08966800 24.18545400 22.37026800 20.97708600 20.17501300 19.96290900 20.69781700 21.06010700 21.45679600 21.44008100 14.19297600 14.41397500 15.08192300 15.27515600 16.42525500 16.74704100 16.27818900 17.48509400 17.31389400 18.47436900 17.57005700 17.83797400 16.62362400 18.60540400 18.10426800 19.18796000 19.51129100 22.42734100 23.96359300 20.11690600 14.67268600 14.79068700 20.39850400 20.68212400

24.72329800 24.87745000 25.17327500 26.51531900 24.57708900 25.30469400 22.22741800 20.98515700 20.08076400 20.76473400 20.33449800 19.20853300 21.47494800 22.77783600 23.72307700 23.15302700 22.10274500 20.76811500 22.56189100 23.12079800 25.39634300 24.72527400 23.52989500 24.59730800 26.38723700 19.07205600 21.27888400 24.56676300 23.38574000 29.49642800 29.95991500 28.41310700 28.47111700 28.54855100 29.58814700 28.36961800 27.57716800 27.35363400 28.02228200 26.23937500 26.42515900 25.70763900 25.32343600 24.53249200 25.88873300 24.66403200 24.79301200 24.44368300 23.11076000 26.97087400 26.13448300 28.57232300 28.15884200

36.82695500 35.41280600 37.64240500 37.71371700 37.19781300 36.14558100 34.54459500 35.14455200 34.11112600 32.89543200 31.54595900 31.07650100 30.70198000 31.10451500 30.14279600 32.34017200 33.18999700 36.35147600 36.69860300 38.11470200 37.11445300 38.62562900 36.91144900 38.06489500 36.27950500 34.21793000 29.72948000 30.50853900 29.27854300 34.43032200 35.30550200 34.02071400 32.93939000 34.74425600 34.64219100 35.81023200 34.22881800 33.17284400 34.32293700 34.96153100 36.00581200 34.98347700 34.28966900 33.72687400 33.54848800 35.21714700 33.66025500 35.07046600 34.98278900 34.21671400 33.35881800 32.30152600 31.42447400

C H C H H C O O C O

21.42462000 21.03917900 21.79326900 22.43493400 20.87914400 22.40893900 22.62628000 22.69297800 22.64526900 22.75545000

28.55185400 28.98350700 27.11192200 27.00240200 26.58649800 26.35765100 26.87389800 25.08420200 29.39707400 30.16204900

33.33796800 34.27579800 33.71363200 34.60025800 33.99460300 32.55658200 31.47625900 32.76270100 33.02388300 32.10476300

R – glycosylase reaction with hOGG1(D268N) O 25.21285800 23.16043900 35.89366200 C 24.06636100 23.16866900 36.72160300 C 23.38824100 24.50021200 36.44060200 O 22.89062800 24.50953700 35.06867000 C 22.17501900 24.94677800 37.27145600 O 22.16955900 26.28718400 37.34523100 C 20.97775700 24.44002700 36.46796800 C 21.50261200 24.38677000 35.02956400 N 21.04142300 23.18394100 34.32354100 C 21.05463000 21.83580700 34.80428900 N 20.82050400 21.06438600 33.70070100 C 20.74073100 21.85556300 32.55764500 C 20.51834500 21.52722600 31.18831400 O 20.27835000 20.44012200 30.69389200 N 20.61670600 22.70166400 30.40360700 C 20.85289900 23.96463700 30.86828700 N 20.97188800 24.94452400 29.95752000 N 20.92455300 24.25796800 32.15829100 C 20.91710100 23.15698500 32.93263100 O 21.17713700 21.47982200 35.96099500 H 23.38860900 22.34941600 36.44531400 H 24.27848800 23.08934200 37.78813300 H 24.15241000 25.27651800 36.49717400 H 22.20205000 24.46178500 38.25256000 H 20.69440200 23.44486900 36.80244400 H 20.13877700 25.12829400 36.60011900 H 21.12509300 25.20195700 34.39920200 H 20.72158400 20.04825500 33.73504500 H 20.63625300 22.56756000 29.38854200 H 21.38927800 25.81494800 30.27830000 H 21.10268400 24.68248400 28.98707200 N 13.93696200 29.72199700 34.29503900 H 14.10023400 30.05870100 35.24019900 C 14.81915300 28.64069100 33.86873800 H 15.03620500 28.74572500 32.79387300 C 16.15243400 28.70358700 34.61605400 H 16.51408400 29.73129200 34.54172500 H 15.99300800 28.50424000 35.67628600 C 17.20974500 27.72191500 34.09907700 H 17.19164300 27.67445700 33.00904200 H 18.19348200 28.10425000 34.38860100

C H H C H H N H H H C O N H C H C H H C O N C O H H

17.11184200 16.92089100 16.29188900 18.40713600 18.71735100 19.22101400 18.27044400 17.66907000 19.19418300 17.82959100 14.34824000 14.42777800 20.47583500 20.87405700 21.34361000 20.83915200 21.72671700 22.21302000 20.77456700 22.50952100 22.35124800 23.35503400 22.53429200 22.66480500 23.72288300 23.34719100

26.28630800 26.29470900 25.74347800 25.55022100 25.60131900 25.94013500 24.09417100 23.61834400 23.61794600 23.97872400 27.20336600 26.40818500 29.33681600 28.90567500 29.28862200 29.66445700 27.82269500 27.69684600 27.28328100 27.11382800 27.41219100 26.15517800 30.21354300 31.06246400 25.55742000 25.75690400

34.62836400 35.70763000 34.15788600 34.34255000 33.29428600 34.95415800 34.68174400 33.99984000 34.66887700 35.63072900 33.99108500 33.08620700 32.02932500 31.20947400 33.18318300 34.08819500 33.52793700 34.50646100 33.59739600 32.42666000 31.23610100 32.82272200 33.06009800 32.22502900 32.09240900 33.76283500

TS1 – glycosylase reaction with hOGG1(D268N) O 25.09956000 23.23186300 35.90680100 C 23.96820200 23.25206300 36.73748600 C 23.45196900 24.68046100 36.67328700 O 22.98785200 24.90959400 35.24251100 C 22.19731900 25.12162800 37.43435100 O 22.19398400 26.45805400 37.51106600 C 21.07384900 24.66758100 36.49924900 C 21.72284000 24.86086300 35.16850000 N 20.69820400 23.02167400 34.26139900 C 20.70071900 21.68123700 34.74328200 N 20.71637400 20.86638400 33.64833900 C 20.69140900 21.64581400 32.48588300 C 20.47763000 21.31284500 31.11623100 O 20.26178400 20.22430600 30.61220900 N 20.52077100 22.49620700 30.33718900 C 20.68361400 23.77083000 30.80157100 N 20.77926700 24.74807400 29.88131400 N 20.70528800 24.06354300 32.09242500 C 20.71024400 22.95705100 32.86531200 O 20.65045700 21.38488300 35.92693500 H 23.19949700 22.56562000 36.35084200 H 24.15773200 23.00200300 37.78313200 H 24.26530600 25.38995200 36.79495100 H 22.13840300 24.60800700 38.39704800 H 20.85893900 23.60202300 36.63906400

H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O N C O H H

20.15658300 21.29777700 20.62506900 20.52427700 21.12819800 20.94859800 13.95096900 14.10819100 14.83476500 15.04710400 16.17015300 16.52092900 16.02148100 17.21977500 17.19437500 18.20890400 17.12476700 16.92873200 16.30924400 18.40839800 18.67411800 19.23486100 18.34813400 17.74380400 19.74846500 17.92443000 14.35560800 14.40180900 20.50095600 20.87513800 21.37071300 20.86534900 21.76297900 22.30851200 20.80788300 22.46015000 22.20910800 23.31715800 22.55298600 22.68972600 23.65238800 23.53139000

25.24376000 25.19240800 19.84934200 22.36484100 25.64730700 24.48021200 29.71842000 30.04249100 28.63899500 28.74647400 28.71488100 29.74704800 28.50793600 27.73897900 27.71355400 28.11787100 26.30408700 26.30034600 25.75690300 25.54711500 25.58554500 26.02382200 24.13710500 23.62956700 23.51321700 24.06258800 27.20275500 26.42548000 29.35661500 28.91612900 29.27590800 29.61586300 27.79956200 27.66352200 27.27528300 27.06832600 27.31937000 26.09136100 30.21453900 31.07640400 25.48011000 25.84147800

36.62658300 34.22381800 33.69578200 29.32451400 30.19536100 28.91648100 34.28937400 35.23985000 33.86238800 32.78673700 34.59979600 34.52412900 35.66129900 34.06506500 32.97234900 34.34678200 34.57742700 35.65577300 34.10484300 34.29188900 33.22822200 34.83487600 34.69299200 34.04559600 34.57436900 35.63346000 33.97658900 33.05546600 31.90420500 31.07802700 33.04961200 33.96806300 33.35069900 34.29733600 33.47901700 32.20447600 31.02520300 32.55064200 32.95754900 32.13821600 31.81575100 33.50490800

IS1 – glycosylase reaction with hOGG1(D268N) O 25.14752300 23.19485700 35.95368900 C 24.01552700 23.20502800 36.78255500 C 23.45669000 24.61614900 36.69610600 O 22.97206200 24.79965900 35.25794500 C 22.19641700 25.04011000 37.45674700 O 22.19023400 26.37602600 37.53252900 C 21.07362200 24.57434100 36.53086900 C 21.71617700 24.74286100 35.20125800 N 20.53050100 22.70108600 34.37627400

C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O

20.50688300 20.63345300 20.68313200 20.60822400 20.47570800 20.67859500 20.75685400 20.88814600 20.67305300 20.62626400 20.37737000 23.26668600 24.20994900 24.25312100 22.15217400 20.84514800 20.15640200 21.27159500 20.57395500 20.75278900 21.15193700 21.07702700 13.92633700 14.08854600 14.81001700 15.01699900 16.14621800 16.49825800 15.98767800 17.20125700 17.17245900 18.18717800 17.12162000 16.92139900 16.31060800 18.41449500 18.67966100 19.23494300 18.36419700 17.68749100 19.74244100 18.00271200 14.34265000 14.41249300 20.48325900 20.84141400 21.35063000 20.84787300 21.72911500 22.30224600 20.77165800 22.37768100 22.08714700

21.38247000 20.55710700 21.32291600 20.98169600 19.88961500 22.16565000 23.44886900 24.42007900 23.74486500 22.63917500 21.10238100 22.48714200 22.98455300 25.34857500 24.52583200 23.51072400 25.15099500 24.91386800 19.53779700 22.02024600 25.34695200 24.15239500 29.75538300 30.08610400 28.67478500 28.77475600 28.76569900 29.79464500 28.59470100 27.76758800 27.67293800 28.17076200 26.36784800 26.43422500 25.78499300 25.59829300 25.56031500 26.12517000 24.22219300 23.70189300 23.32692200 24.22851800 27.23610600 26.44381900 29.36936700 28.90937700 29.26755500 29.60775900 27.78505100 27.65089300 27.27641600 27.02044000 27.23895100

34.87858100 33.79727800 32.62659400 31.24590500 30.72069100 30.46716400 30.93058200 30.00961600 32.21815100 32.99414700 36.06212500 36.41284600 37.83376800 36.79355400 38.41918200 36.68082100 36.64633300 34.21793900 33.85250100 29.46167400 30.32146300 29.04542000 34.26913900 35.21640800 33.84305500 32.76563300 34.57901600 34.47420500 35.64398000 34.09390800 33.00426400 34.35071800 34.70022800 35.77613800 34.26108600 34.47694900 33.41239000 34.98543500 34.97856700 34.42072100 34.74079900 35.94741900 33.97219600 33.06482400 31.94389400 31.12087700 33.08971700 34.00974300 33.39001300 34.32025900 33.55817100 32.23544800 31.06069100

N C O H H

23.23052200 22.53698000 22.67483800 23.54742800 23.48921200

26.03389600 30.20337500 31.06469500 25.41136900 25.81203800

32.57500600 33.00224900 32.18255400 31.84133400 33.52389500

R – glycosylase reaction with WT hOGG1 where Asp268 was protonated; Asp268(CO2H) O 25.19043400 23.24445900 35.89644200 C 24.04756500 23.25265800 36.72474900 C 23.39272100 24.60373100 36.48925900 O 22.88307300 24.66645400 35.11751400 C 22.18010000 25.03512500 37.32764400 O 22.17435800 26.37480800 37.40306900 C 20.97925800 24.52727900 36.52292500 C 21.50093300 24.45970300 35.08437100 N 21.09413800 23.23277600 34.39583700 C 21.02008100 21.89803800 34.89094300 N 20.68805200 21.13725600 33.80306400 C 20.65468600 21.91930000 32.64995600 C 20.37975300 21.60589600 31.28706100 O 20.03466300 20.54447500 30.79884900 N 20.56179300 22.76685300 30.49519100 C 20.89752800 24.01152000 30.94950200 N 21.08222200 24.97930200 30.03691300 N 20.99810100 24.30251200 32.23698900 C 20.93518100 23.20715000 33.01121400 O 21.15184800 21.54345000 36.04785300 H 23.35510900 22.45263700 36.42749900 H 24.25801700 23.13867100 37.78902700 H 24.16546700 25.36878600 36.56188700 H 22.21167900 24.54620000 38.30603100 H 20.68229400 23.53917000 36.86548100 H 20.14679500 25.22554000 36.64517600 H 21.09522600 25.24152200 34.43692300 H 20.57997800 20.12323100 33.83760000 H 20.56630200 22.61538600 29.48597700 H 21.63015500 25.77331400 30.35063800 H 21.16967500 24.70025100 29.06209800 N 14.02579600 29.70310700 34.32012700 H 14.19364700 30.05593500 35.25848200 C 14.90791100 28.62170000 33.89536800 H 15.14832300 28.74153400 32.82622400 C 16.22893100 28.64743500 34.66987800 H 16.60801800 29.66985000 34.62161000 H 16.04786800 28.43443300 35.72423400 C 17.28028900 27.65082800 34.15654500 H 17.27244400 27.61153700 33.06921700 H 18.26723000 27.99904000 34.47354200 C 17.13750800 26.20613900 34.65404100 H 16.97819500 26.19427600 35.73823400 H 16.28818200 25.70546600 34.19182000 C 18.39243800 25.42985000 34.30987700 H 18.67365900 25.49208900 33.25489400

H N H H H C O N H C H C H H C O O C O H

19.23450100 18.23083600 17.64324100 19.15529600 17.78643600 14.41844600 14.48071900 20.52455700 20.96949500 21.41173200 20.90466700 21.84373000 22.35167400 20.91318700 22.65453100 22.66467300 23.35095100 22.58702300 22.67314700 23.24435700

25.78464100 23.97114800 23.48642300 23.50559800 23.85837900 27.19054700 26.41277200 29.19506700 28.83788600 29.28723900 29.70418600 27.85254800 27.77459500 27.28413700 27.16333400 27.59862300 26.09670400 30.21800500 31.02050300 25.72259700

34.90111900 34.63599300 33.94996500 34.64715200 35.58566700 33.99481000 33.07460300 32.21838500 31.38873500 33.35603500 34.23954700 33.77567300 34.74848700 33.88261900 32.67877300 31.53881400 32.98555200 33.14343100 32.25828200 33.89950400

TS1 – glycosylase reaction with WT hOGG1 where Asp268 was protonated; Asp268(CO2H) O 25.08510300 23.26755600 35.92915700 C 23.95717200 23.28090100 36.76052000 C 23.44112000 24.70807000 36.71914200 O 22.96117900 24.94423200 35.28848700 C 22.18954100 25.13229300 37.49072100 O 22.18669100 26.46969700 37.57046800 C 21.05681100 24.69070300 36.55185400 C 21.69464700 24.88405700 35.21469800 N 20.65526500 23.00158300 34.34822300 C 20.69710600 21.67464300 34.80945200 N 20.63842900 20.84889000 33.71395200 C 20.61070800 21.63188400 32.55829700 C 20.36145900 21.33106900 31.19136600 O 20.06996500 20.27130300 30.66069500 N 20.47568800 22.52438900 30.42887100 C 20.69688000 23.78091500 30.91075200 N 20.84892400 24.76911800 30.00120800 N 20.71087600 24.05710600 32.20217900 C 20.67867800 22.93928800 32.96689800 O 20.73943700 21.35570200 35.99538000 H 23.18902700 22.59438400 36.37240700 H 24.15080900 23.02528600 37.80494000 H 24.25329100 25.41827500 36.84241300 H 22.13563700 24.60653500 38.44747000 H 20.84019700 23.62194800 36.65392600 H 20.14573700 25.27378600 36.70059000 H 21.27034400 25.13228600 34.24356000 H 20.57717500 19.83196700 33.75752900 H 20.49260000 22.39337300 29.41886400 H 21.37327800 25.57358000 30.31781800 H 20.99614800 24.48443300 29.03363200

N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O H

14.01465900 14.18336600 14.89672200 15.12788000 16.22251600 16.59654800 16.05343000 17.26133200 17.22964600 18.25586700 17.13548700 16.96681000 16.29723900 18.40670400 18.69648700 19.22100600 18.32122800 17.73878600 19.43787300 17.88789100 14.40812300 14.45555600 20.50709100 20.94086400 21.39938300 20.89331100 21.85097900 22.37675300 20.92440900 22.63364000 22.60790400 23.32707000 22.57343100 22.67133700 23.41043300

29.72146800 30.07216000 28.64055800 28.75704400 28.67641700 29.70062200 28.46355400 27.67857900 27.63251700 28.02756000 26.23972800 26.23676000 25.71679700 25.47376000 25.53540200 25.89873300 24.03845700 23.52940500 23.54629400 23.92914500 27.20879200 26.43643500 29.22798500 28.88170300 29.30590100 29.69213900 27.86354600 27.76518900 27.28808600 27.20730800 27.63526100 26.10785500 30.24555800 31.05631600 25.89505000

34.29604600 35.23500000 33.87032000 32.79885500 34.63089900 34.57613700 35.68814400 34.10102600 33.01416700 34.39425900 34.61260500 35.69519400 34.15464200 34.29697000 33.24321800 34.89033400 34.65101200 33.98305800 34.61733200 35.59261200 33.97448600 33.04996300 32.14663700 31.30661400 33.27691400 34.17467700 33.64867800 34.61192200 33.75484500 32.51357400 31.38069500 32.78099400 33.08948400 32.21547800 33.72339600

IS1 – glycosylase reaction with WT hOGG1 where Asp268 was protonated; Asp268(CO2H) O 25.15046100 23.19880400 35.84671600 C 24.02346600 23.19889800 36.67700400 C 23.39397300 24.57608300 36.55599800 O 22.81210200 24.65111600 35.13671000 C 22.16394300 24.97751200 37.37685200 O 22.15537100 26.31224400 37.45226400 C 20.99810700 24.46962800 36.52367100 C 21.55983200 24.53958100 35.15847800 N 19.97534200 22.26707900 34.55203100 C 19.65858700 20.96728700 34.97660500 N 19.77862900 20.16692600 33.87669600 C 20.12529800 20.94190600 32.76735300 C 20.19083000 20.65549500 31.37616000 O 19.99726800 19.60739200 30.78444800 N 20.51095100 21.84144100 30.66365000 C 20.68066200 23.08984700 31.18507800

N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H H C O N H C H C H H C O O C O H

20.99794600 20.49667800 20.21666300 19.34752400 23.29963300 24.22894200 24.16471200 22.18710900 20.75079300 20.08637100 21.05602300 19.73649600 20.70347000 21.49333100 21.20071400 13.96050300 14.13392900 14.84335700 15.04933300 16.18059000 16.53092000 16.02142600 17.23112200 17.19203600 18.22329300 17.11202400 16.91472800 16.28143300 18.35460600 18.59974900 19.21588100 18.21297700 17.45549100 19.39361400 17.91561200 14.37076800 14.42712700 20.47932900 20.91390400 21.37071200 20.87207100 21.79317700 22.35415200 20.86501800 22.53009200 22.64997400 22.99914600 22.56093400 22.67045400 23.08144000

24.08306500 23.35002400 22.23980700 20.66102100 22.43823500 23.02717600 25.34327500 24.46928800 23.42096700 25.05460600 24.51828700 19.15008600 21.69329400 24.86826100 23.83856300 29.84656700 30.19259900 28.76600700 28.86639200 28.84963800 29.87910700 28.67090500 27.84186200 27.71276200 28.22090100 26.45647400 26.55271800 25.89494000 25.60505700 25.48522600 26.10570800 24.27186800 23.78287500 23.05990400 24.36879200 27.32970400 26.55627800 29.39810900 29.09332900 29.44538000 29.83745900 27.99382500 27.88444500 27.42907700 27.33477200 27.85597400 26.09366000 30.36541600 31.17483800 25.83470500

30.33002800 32.47015400 33.19162500 36.11972400 36.34148100 37.73572200 36.57626200 38.34200400 36.73393400 36.63857700 34.18057500 33.92248900 29.67386900 30.72519900 29.35754600 34.29506800 35.23558100 33.86796400 32.79093000 34.60190500 34.50817500 35.66400700 34.12109900 33.03968200 34.38221200 34.75789000 35.83226100 34.32982700 34.56164000 33.49861400 35.02836100 35.15237500 34.67696500 34.92994700 36.13710900 33.98476200 33.06010100 32.14682100 31.29122600 33.28177800 34.18064900 33.65069100 34.59293500 33.79785300 32.49400200 31.41379200 32.67555500 33.09457300 32.22100100 33.60314900

R – SN1 base-excision reaction with WT hOGG1; Asp268(CO2-), Lys249 (NH2) O 25.30852200 23.07720800 36.27216000

C C O C O C C N C N C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H C O N H

24.14995400 23.34116100 22.93509100 22.05730400 22.04725900 20.92392300 21.56638500 21.30871400 21.38760800 21.16039300 20.94702500 20.74462000 20.65272100 20.64024500 20.76796800 20.68179400 20.93671200 21.05236200 21.58965700 23.55363700 24.35068200 24.01183400 22.05517000 20.53093000 20.10477500 21.15152100 21.12964400 20.57198500 20.89386400 20.82292200 14.32944800 14.52911500 15.23476200 15.44301400 16.57094400 16.82356400 16.42082200 17.69401600 17.52709000 18.63662300 17.87799700 18.68804900 16.98212500 18.20355100 17.33875200 19.07410600 18.50682300 18.66303100 17.74241800 14.86122300 15.01283100 20.46828500 20.81737400

23.08749800 24.26915700 24.05827700 24.67602500 26.01950600 24.10351600 23.80332400 22.43725200 21.20592100 20.24755500 20.85578100 20.36337200 19.20699000 21.44040300 22.77545700 23.65206300 23.20135200 22.21707900 21.00890200 22.17546000 23.21866200 25.13256900 24.19709100 23.18631500 24.81502000 24.42815500 19.25368000 21.16775500 24.63891300 23.30234300 29.23371400 29.66786000 28.15935100 28.25027300 28.35902600 29.41679600 28.18846800 27.46691900 27.30616000 28.01481600 26.09843900 26.14955100 25.80608600 25.01186300 24.96044000 25.29933900 23.76340700 23.01993900 23.48487400 26.71526300 25.85190200 29.15462900 28.57950400

37.09007500 36.58080800 35.21983300 37.34190900 37.41883700 36.47877500 35.11824000 34.61905600 35.28357100 34.31569400 33.07713700 31.76724500 31.34568600 30.86071200 31.15750200 30.14819200 32.39128600 33.28864400 36.47240500 36.96101500 38.15516900 36.57935300 38.33069100 36.90650100 36.37830100 34.32466500 34.50333500 29.88664900 30.40914200 29.20646600 34.24669100 35.13907600 33.82388500 32.74765400 34.56594400 34.44617100 35.63891700 34.03177100 32.95970600 34.10162200 34.69949100 35.43447500 35.26074200 33.67863100 32.99054400 33.07021700 34.35402000 33.68145100 34.97000400 34.01839800 33.19449800 31.46395600 30.71159000

C H C H H C O O C O

21.23204500 20.69469700 21.42089200 22.17277100 20.47417300 21.76565500 22.75892600 20.91216400 22.47682900 22.69709200

29.01954600 29.47716100 27.52854500 27.29808300 27.13560700 26.84038300 27.30976400 26.02173400 29.86716700 30.80628700

32.70428800 33.55635800 33.06317200 33.83017300 33.43779600 31.72826300 31.13340600 31.31213800 32.67557600 31.95891300

LRC3 – Ribose-protonated base-excision reaction with WT hOGG1; Asp268 (CO2H), Lys249 (NH2) O 25.08682100 23.12975400 36.33635300 C 23.93557100 23.15001200 37.15340700 C 23.27307900 24.48436100 36.86053700 O 22.81516600 24.55850900 35.47902500 C 22.01798600 24.87184200 37.64862300 O 22.00887100 26.21328800 37.72394900 C 20.87160400 24.30023100 36.80423700 C 21.45117900 24.18737700 35.39111200 N 21.27698200 22.90230700 34.73360200 C 21.06351000 21.62506900 35.27979100 N 20.71590100 20.82883200 34.21264400 C 20.67113800 21.58155800 33.04078700 C 20.39444900 21.25147200 31.68661100 O 20.09039500 20.17709900 31.18288700 N 20.51791100 22.41546700 30.88260100 C 20.87047300 23.65738900 31.32118500 N 20.96178800 24.64886300 30.39160600 N 21.07871500 23.94565100 32.58313400 C 21.00814600 22.86669500 33.38675000 O 21.16845500 21.28836700 36.44930000 H 23.25078100 22.33604600 36.88027100 H 24.13109600 23.08604700 38.22833400 H 24.02308600 25.26678100 36.97100800 H 22.03671700 24.38928300 38.63309300 H 20.58228500 23.31707900 37.16310500 H 20.00581600 24.96405700 36.83387200 H 20.98811200 24.88959900 34.69883700 H 20.50398800 19.84082700 34.30291100 H 20.45888400 22.26830500 29.87160200 H 21.54979800 25.42045000 30.69254500 H 21.11209200 24.34782900 29.43685400 N 13.96120100 29.43779500 34.34413300 H 14.14354600 29.81619300 35.26752400 C 14.85658600 28.35732800 33.92162400 H 15.08803800 28.46911000 32.85146600 C 16.17292500 28.44473000 34.70193300 H 16.51412100 29.47987000 34.62643200 H 15.98579400 28.25859000 35.76143100 C 17.25795700 27.46694500 34.24123100 H 17.30047500 27.42041000 33.15463300

H C H H C H H N H H C O N H C H C H H C O O C O H

18.22310400 17.15151000 16.91217700 16.36382700 18.47554900 18.80613600 19.23189000 18.49599100 18.03775000 17.98539500 14.38929100 14.40119800 20.50564000 20.91351900 21.39958000 20.92834700 21.76656800 22.24399900 20.82240600 22.60156300 22.62514700 23.32123800 22.61674600 22.72563300 23.16964600

27.84525800 26.02226600 26.00143100 25.46875000 25.30280900 25.45107700 25.79991100 23.87996900 23.35926500 23.68257500 26.93011900 26.15608900 29.00143700 28.56051300 29.05255300 29.53280600 27.61942000 27.58102300 27.07480000 26.86362000 27.22857300 25.84755600 29.91124300 30.68450200 25.49405300

34.59061900 34.73334200 35.80406200 34.22071600 34.49090200 33.45322600 35.11572200 34.77176600 34.02972900 35.63797500 34.03957000 33.11933700 32.29182400 31.48332800 33.42955300 34.30004800 33.91783400 34.90670700 34.02644800 32.88801700 31.71924700 33.28526800 33.15959400 32.24855700 34.21241600

RP1 maximum – Ribose-protonated base-excision reaction with WT hOGG1; Asp268 (CO 2H), Lys249 (NH2) O 25.21895900 23.02065000 36.50659900 C 24.05980600 23.01838800 37.30843200 C 23.15556300 24.03558000 36.63030600 O 22.77146900 23.57952600 35.33026500 C 21.88404300 24.43713800 37.42020400 O 21.87297200 25.77658000 37.49332600 C 20.61223800 23.82710500 36.79651800 C 20.69119900 23.66388600 35.31733700 N 20.54713800 22.48061500 34.66207600 C 20.44370000 21.14173000 35.14103400 N 20.42565800 20.37507600 34.00786700 C 20.49323100 21.17547300 32.85774000 C 20.49930200 20.90974700 31.45538600 O 20.40734000 19.84105100 30.86904400 N 20.64675000 22.12677000 30.73998600 C 20.73808600 23.38270600 31.27592200 N 20.95985400 24.43088600 30.46736400 N 20.58663400 23.60358100 32.56840400 C 20.54490900 22.47429500 33.27267100 O 20.39457700 20.79297600 36.30276500 H 23.54122700 22.04302300 37.31194000 H 24.21578700 23.31372400 38.34920600 H 23.74556600 24.94811400 36.52106800 H 21.95819600 23.98769900 38.42184800 H 20.41004700 22.85253800 37.23073700 H 19.79568100 24.50565900 37.05106800

H H H H H N H C H C H H C H H C H H C H H N H H C O N H C H C H H C O O C O H

20.86245400 20.41923900 20.78657300 21.33856000 21.28941300 13.94681100 14.14914500 14.84498700 15.05128100 16.17357700 16.48450000 16.00370300 17.27962300 17.31207200 18.23579800 17.22230500 16.90795800 16.50751200 18.59243700 19.05711600 19.26860700 18.56475500 18.27729200 17.98025200 14.43335100 14.50844400 20.51209600 20.81594700 21.39087500 20.95345800 21.61478900 22.23872700 20.63033900 22.09522800 21.94628400 22.53431500 22.63878600 22.79270900 22.95634000

24.50944800 19.35909000 22.03865500 25.26766100 24.23893200 29.60232000 30.00853700 28.52463700 28.61789300 28.67788900 29.71731600 28.52636100 27.71406100 27.58303900 28.14529000 26.31295000 26.35170900 25.67576000 25.65662600 25.83945000 26.10554300 24.22366800 23.72541100 23.93632700 27.08732500 26.28329000 29.23717400 28.55035500 29.26665000 29.84183300 27.84467900 27.82769900 27.52773400 26.70038400 26.85529000 25.68534200 30.06496500 30.82412900 24.30409800

34.66044900 34.05254800 29.72638900 30.93807700 29.53144700 34.32459900 35.23244700 33.89991200 32.82299700 34.64745800 34.52545500 35.71249000 34.22796500 33.14831500 34.53514600 34.83175300 35.88208800 34.30843100 34.73737900 33.75870700 35.48296300 34.94272100 34.10677900 35.73745600 34.05917800 33.16517800 32.27850100 31.59938000 33.43047500 34.26515300 34.02550400 34.92707600 34.38542800 33.08568600 31.84320900 33.66994800 33.14320000 32.22542700 34.68970700

RP1 – Ribose-protonated base-excision reaction with WT hOGG1; Asp268 (CO 2H), Lys249 (NH2) O 25.23493300 23.12220900 36.53067900 C 24.07864600 23.12884400 37.33094400 C 23.07703200 23.90712600 36.48820300 O 22.79529000 23.15788700 35.31812300 C 21.80674400 24.32343100 37.29785300 O 21.79608600 25.66554900 37.37243400 C 20.49863500 23.69317200 36.79742600 C 20.22236100 23.68669800 35.30219800 N 20.19274900 22.40575900 34.65401600 C 20.29354600 21.08139000 35.13919200 N 20.46224100 20.31215600 34.01535900

C C O N C N N C O H H H H H H H H H H H N H C H C H H C H H C H H C H H N H H C O N H C H C H H C O O C O

20.52068600 20.67871200 20.73208500 20.78751600 20.71020600 20.98453600 20.35030500 20.36245800 20.20215200 23.65866600 24.19214700 23.56271200 21.92614800 20.48256800 19.69129100 20.88927700 20.56506400 21.04285300 21.34395700 21.41095600 14.11249700 14.32001500 15.01051300 15.21542200 16.33701700 16.63768800 16.15212900 17.46620200 17.50237900 18.40866200 17.45125400 17.10651600 16.76226300 18.85610000 19.37612000 19.48896800 18.82601100 18.52593400 18.15532100 14.63141800 14.77526300 20.69447700 20.99613000 21.58789300 21.17330300 21.79901800 22.52390800 20.85484200 22.07452900 22.02949700 22.25491500 22.83605500 22.97903200

21.10960500 20.86260900 19.80068600 22.10022700 23.35121200 24.42297900 23.54099500 22.40085300 20.70911400 22.12275600 23.61292900 24.84816000 23.88234900 22.65410900 24.22657300 24.34962600 19.30252400 22.03465800 25.24764600 24.23972200 29.55777700 29.99137100 28.47921100 28.55850300 28.65764300 29.69704000 28.53356600 27.70524900 27.52632900 28.17449900 26.32885200 26.40853600 25.65183300 25.74518200 25.95054100 26.11360900 24.26592800 23.88762500 23.94073800 27.03963100 26.19954100 29.23449100 28.54953700 29.27558300 29.87366700 27.86031800 27.81049600 27.61673700 26.64814300 26.82453200 25.57092900 30.05434000 30.79285900

32.86860900 31.47549900 30.87166100 30.78201600 31.32582000 30.56059600 32.58327100 33.28665800 36.29488600 37.50905200 38.30554400 36.21434000 38.30242600 37.11723200 37.30751800 34.74227300 34.07477100 29.78945400 31.07181500 29.66218500 34.34478600 35.23886100 33.92422400 32.84577800 34.67488500 34.53043100 35.74074400 34.28934600 33.21567000 34.57660000 34.96526200 36.00230300 34.45360900 34.92612600 33.98373400 35.73949500 35.03340800 34.12983000 35.76080400 34.11970800 33.26747500 32.33890200 31.65650500 33.47765500 34.30699900 34.09333600 34.91519500 34.59139100 33.15152400 31.90185100 33.75690800 33.14722100 32.21052500

H

22.90957300 23.76034700 34.56669000

R – glycosylase reaction with WT hOGG1 (calculated with completely explicitly hydrated 2NOZ structural model) N 42.09800000 59.03100000 47.69400000 C 42.99800000 57.94400000 47.25800000 C 42.64400000 56.50000000 47.47100000 O 42.80100000 55.65100000 46.62700000 C 44.36500000 58.14900000 47.93800000 C 45.48400000 57.25500000 47.40400000 C 45.60600000 55.87600000 48.04600000 C 46.87100000 55.19400000 47.55400000 N 46.97100000 53.83100000 48.16800000 H 42.19200000 59.25300000 48.68400000 H 43.17100000 58.01200000 46.17600000 H 44.27100000 57.99400000 49.01400000 H 44.62200000 59.20200000 47.79500000 H 46.43200000 57.75900000 47.58900000 H 45.38800000 57.14200000 46.31900000 H 44.75100000 55.23800000 47.81400000 H 45.64300000 55.99700000 49.13300000 H 47.77900000 55.73500000 47.83300000 H 46.89800000 55.05300000 46.47300000 H 47.89500000 53.38200000 47.93200000 H 46.89700000 53.92700000 49.20000000 H 46.21100000 53.23600000 47.83100000 N 49.46500000 60.34300000 45.65900000 C 50.34400000 59.98100000 46.78400000 C 51.64300000 60.68500000 46.52700000 O 51.83200000 61.46300000 45.63400000 C 50.50900000 58.49800000 47.16300000 C 51.13000000 57.64400000 46.03500000 O 51.62300000 58.30200000 45.08200000 O 51.07400000 56.40900000 46.19400000 H 49.95700000 60.20900000 44.78400000 H 49.98600000 60.51100000 47.68200000 H 49.53800000 58.07000000 47.43000000 H 51.10400000 58.37800000 48.07900000 O 54.01800000 52.97800000 49.34200000 C 52.86100000 53.01100000 50.16300000 C 52.08400000 54.23900000 49.65900000 O 51.59500000 53.92000000 48.34200000 C 50.22900000 54.14400000 48.20500000 N 49.58700000 52.95800000 47.56600000 C 49.80600000 51.64200000 48.10300000 O 50.00300000 51.37400000 49.27200000 N 49.66900000 50.78300000 47.05100000 C 49.49900000 51.49600000 45.86600000 C 49.44500000 51.07000000 44.51100000 O 49.51100000 49.93800000 44.04100000 N 49.32000000 52.18800000 43.67300000 C 49.23400000 53.49500000 44.07500000 N 49.08200000 54.39000000 43.08300000 N 49.24000000 53.86200000 45.34200000

C C C O H H H H H H H H H H H

49.45800000 50.87200000 49.66700000 50.87500000 53.08500000 52.24400000 52.80400000 50.06600000 49.94000000 49.40700000 49.32900000 49.23400000 50.84500000 49.27300000 48.91200000

52.83200000 54.78600000 54.34500000 56.12900000 53.10300000 52.11800000 55.05800000 54.98300000 49.79400000 52.00500000 54.07800000 55.38900000 54.29900000 53.40300000 55.13600000

46.17200000 50.46200000 49.62100000 50.54100000 51.22400000 50.00400000 49.56900000 47.51100000 47.10400000 42.67400000 42.15300000 43.29500000 51.44600000 50.00400000 49.66400000

TS1 – glycosylase reaction with WT hOGG1 (calculated with explicitly hydrated 2NOZ structural model) N C C O C C C C N H H H H H H H H H H H H H N C C O C C O O H H H H O

42.14800000 43.04700000 42.67700000 42.81800000 44.41100000 45.53400000 45.64100000 46.91800000 47.03200000 42.23900000 43.22900000 44.30600000 44.67700000 46.48100000 45.46700000 44.79300000 45.64900000 47.80800000 46.97000000 48.08200000 46.93300000 46.27400000 49.43500000 50.31300000 51.61300000 51.80300000 50.46400000 51.04500000 51.58700000 50.90900000 49.92300000 49.96200000 49.49200000 51.07900000 53.90200000

58.97500000 57.88500000 56.44300000 55.59600000 58.07100000 57.17900000 55.77500000 55.09600000 53.72600000 59.19500000 57.95500000 57.90600000 59.12500000 57.67100000 57.11200000 55.14400000 55.85300000 55.65800000 54.99900000 53.18400000 53.77400000 53.14800000 60.27400000 59.91800000 60.61700000 61.38700000 58.44000000 57.55100000 58.16200000 56.31800000 60.13800000 60.46100000 58.03400000 58.32700000 52.83100000

47.70100000 47.26600000 47.47100000 46.62400000 47.95900000 47.42300000 48.01000000 47.53200000 48.09100000 48.69200000 46.18600000 49.03400000 47.83100000 47.64800000 46.33200000 47.73700000 49.10100000 47.83200000 46.44600000 47.79400000 49.11400000 47.72400000 45.66500000 46.79000000 46.52700000 45.62700000 47.19400000 46.07700000 45.12600000 46.24400000 44.78800000 47.68200000 47.49100000 48.09800000 49.24700000

C C O C N C O N C C O N C N N C C C O H H H H H H H H H H H

52.74500000 52.06700000 51.62600000 50.36400000 49.21800000 49.53500000 49.67800000 49.63100000 49.45500000 49.45700000 49.60000000 49.25300000 49.08800000 48.95200000 49.00400000 49.22500000 50.81000000 49.68400000 50.81000000 52.95900000 52.08500000 52.82400000 50.03500000 49.94100000 49.36000000 49.30500000 49.08000000 50.73800000 49.38300000 48.84400000

52.83700000 54.16300000 54.17100000 54.29900000 52.55200000 51.29600000 51.06000000 50.41100000 51.09600000 50.68100000 49.56300000 51.79400000 53.09200000 53.98700000 53.44500000 52.41500000 54.65700000 54.23300000 55.99700000 52.80700000 51.99400000 54.94300000 54.70800000 49.43900000 51.62100000 53.69000000 54.98200000 54.15100000 53.19900000 54.92100000

50.06300000 49.73700000 48.31500000 48.19400000 47.37200000 47.91600000 49.11500000 46.87300000 45.67300000 44.32000000 43.82700000 43.48600000 43.88700000 42.88400000 45.15200000 45.99700000 50.47700000 49.53600000 50.55600000 51.13100000 49.81300000 49.75400000 47.24100000 46.97100000 42.48900000 41.98100000 43.12200000 51.44600000 49.74100000 49.61600000

IS1 – glycosylase reaction with WT hOGG1 (calculated with explicitly hydrated 2NOZ structural model) N C C O C C C C N H H H H H H H H H H H

42.07300000 42.97400000 42.59600000 42.70100000 44.33200000 45.45200000 45.50500000 46.79000000 46.91500000 42.16000000 43.15800000 44.22400000 44.59700000 46.40000000 45.42700000 44.67100000 45.45200000 47.66200000 46.86000000 48.25900000

58.99300000 57.90600000 56.46500000 55.64100000 58.10000000 57.19500000 55.78800000 55.08000000 53.75800000 59.19600000 57.98400000 57.94000000 59.15300000 57.66700000 57.13700000 55.16700000 55.85300000 55.68100000 54.94700000 53.01200000

47.69800000 47.26000000 47.44700000 46.57400000 47.95700000 47.45100000 48.03300000 47.63300000 48.25200000 48.69300000 46.18000000 49.03100000 47.82200000 47.71500000 46.35600000 47.70300000 49.12700000 47.91800000 46.55000000 47.68600000

H H N C C O C C O O H H H H O C C O C N C O N C C O N C N N C C C O H H H H H H H H H H H

46.82600000 46.14900000 49.44400000 50.32100000 51.61500000 51.79900000 50.49400000 51.15600000 51.66200000 51.12400000 49.93300000 49.95800000 49.52200000 51.07000000 53.93600000 52.78800000 52.21000000 51.92200000 50.69000000 48.97500000 49.33100000 49.40000000 49.58100000 49.39900000 49.56500000 49.88800000 49.30400000 48.98800000 48.87200000 48.77200000 49.01100000 50.87000000 49.87300000 50.86600000 52.98700000 52.06300000 52.98300000 50.38600000 49.95700000 49.51900000 49.31500000 48.93900000 50.69700000 49.60700000 48.96500000

53.86200000 53.16700000 60.20600000 59.85500000 60.57100000 61.34300000 58.38100000 57.51400000 58.12900000 56.27400000 60.09400000 60.38900000 57.94500000 58.28600000 52.87100000 52.90000000 54.30500000 54.40300000 54.45900000 52.38900000 51.18600000 51.00300000 50.30800000 50.95000000 50.53200000 49.44000000 51.60800000 52.88700000 53.77000000 53.24300000 52.24300000 54.77200000 54.34400000 56.11100000 52.74200000 52.14700000 55.05100000 54.58200000 49.36400000 51.44200000 53.49500000 54.76500000 54.24700000 53.27700000 54.94300000

49.26500000 47.93500000 45.67900000 46.80600000 46.54900000 45.64900000 47.20900000 46.12400000 45.16100000 46.33000000 44.80000000 47.70000000 47.46000000 48.14000000 49.28400000 50.10500000 49.89800000 48.43100000 48.19800000 47.20400000 47.81100000 49.02500000 46.79200000 45.56600000 44.22300000 43.76400000 43.35500000 43.72100000 42.70500000 44.96800000 45.83800000 50.51900000 49.43400000 50.60100000 51.16400000 49.76200000 50.05000000 47.16200000 46.92600000 42.37300000 41.83500000 42.96400000 51.46500000 49.53300000 49.41200000

Supporting References 70. D.A. Case, T.A.D., T.E. Cheatham, III, C.L. Simmerling, J. Wang, R.E. Duke, R. Luo, M. Crowley, R.C. Walker, W. Zhang, K.M. Merz, B. Wang, S. Hayik, A. Roitberg, G. Seabra, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, X. Wu, S.R. Brozell, T. Steinbrecher, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak, G. Cui, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, and P.A. Kollman. (2012). University of California, San Francisco. 71. Maestro, v. 9.3, Schroedinger, LLC: New York, 2013. 72. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., et .al. (2009) Gaussian 09, revision B.01; Gaussian, Inc.: Wallingford, CT, 2009. Rutledge, L.R. and Wetmore, S.D. (2011) Modeling the chemical step utilized by human 73. alkyladenine DNA glycosylase: A concerted mechanism aids in selectively excising damaged purines. J. Am. Chem. Soc., 133, 16258-16269. 74. Swiderek, K., Marti, S., Tunon, I., Moliner, V. and Bertran, J. (2015) Peptide bond formation mechanism catalyzed by ribosome. J. Am. Chem. Soc., 137, 12024-12034. 75. Molecular Operating Environment (MOE), C.C.G.I., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2016. 76. Li, H., Robertson, A.D., Jensen, J.H. (2005) Very Fast Empirical Prediction and Rationalization of Protein pKa Values. Proteins, 61, 704-721. 77.Krieger, E. and Vriend, G. (2014) YASARA View-molecular graphics for all devices-from smartphones to workstations. Bioinformatics, 30, 2981-2982. 78.Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., Klein, M. L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys., 79, 926-935. 79.Berendsen, H. J. C., Vanderspoel, D., Vandrunen, R. (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput. Phys. Commun., 91, 43-56. 80.Hess, B., Kutzner, C., van der Spoel, D., Lindahl, E. (2008) GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput., 4, 435-447. 81.Pronk, S., Pall, S., Schulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M. R., Smith, J. C., Kasson, P. M., van der Spoel, D., Hess, B., Lindahl, E. (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics, 29, 845-854. 82.Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., Simmerling, C. (2006) Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins, 65, 712-725. 83.Bayly, C. I., Cieplak, P., Cornell, W. D., Kollman, P. A. (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem., 97, 10269-10280.