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Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 9, 3063-3070, 1 May 2007

doi:10.1529/biophysj.106.092106

Biophysical Theory and Modeling

Differing Conformational Pathways Before and After Chemistry for Insertion of dATP versus dCTP Opposite 8-OxoG in DNA Polymerase β

Yanli Wang^*, Sujatha Reddy^*, William A. Beard^†, Samuel H. Wilson^† and Tamar Schlick^*, ,  

^* Department of Chemistry and Courant Institute of Mathematical Sciences, New York University, New York, New York
^† Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Address reprint requests to T. Schlick.

DNA base and sugar lesions caused by cellular reactive oxygen species contribute to mutagenesis and carcinogenesis 1. One of the most prevalent lesions in the genome is 7,8-dihydro-8-oxoguanine (8-oxoG) 2 (Figure 1b), in which C8 is oxidized to a carbonyl group and N7 is transformed to an –NH– group. The 8-oxoG nucleoside can adopt two glycosidic conformations: the anti conformation when paired with a complementary C (Figure 1c), and the syn conformation when paired with A to form a Hoogsteen basepair (Figure 1d); the latter avoids a clash between the deoxyribose backbone and the 8-oxo group. When it is not repaired, the mismatched 8-oxoG:A basepair can introduce G:C to T:A transversion mutations (i.e., the mispairing of 8-oxoG with A will result in thymine in the next replication cycle). Such mutations contribute significantly to somatic mutations associated with spontaneous cell transformations 3,4,5,6.

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Figure 1

The molecular formulas of guanine (a) and 8-oxoG (b) and the two possible 8-oxoG pairing forms: (c) anti Watson-Crick pairing, with C; and (d) syn Hoogsteen pairing, with A.

High- and low-fidelity DNA polymerases, such as human DNA polymerase β and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), often encounter 8-oxoG7,8,9,10,11 and can bypass it in the replication or repair process. Steady-state kinetic data suggest that pol β prefers dCTP over dATP incorporation by twofold and that dCTP insertion is only threefold less efficient opposite 8-oxoG than opposite a normal dG12.

A partial explanation to the accommodation of 8-oxoG:dCTP comes from the ternary crystal structure of the pol β/substrate complex with 8-oxoG13: the structure shows that the matched 8-oxoG (anti):dCTP basepair is easily tolerated by pol β due to a 184° flip of the template backbone (O3′-P-O5′-C5′), thereby avoiding the clash between O8 and O5′ and O1P of 8-oxoG. The mismatched 8-oxoG (syn):dATP basepair, in contrast, is not as stable as the matched pair in pol β. Crystallography has not captured the syn conformation of 8-oxoG pairing with dATP in pol β but rather captured anti 8-oxoG stacking with dAMP 12.

Besides the crystal structures of pol β 8-oxoG complexes, other DNA polymerases across different families with 8-oxoG at the template position have also been resolved crystallographically. These include RB69 from the B-family 10, T7 DNA polymerase 14,15 and Bacillus fragment (BF) from the A-family 11, and Dpo4 from the Y-family 16. However, none of these wild-type enzyme complexes captured syn 8-oxoG pairing with dATP at the active site. Recently, only by mutating Lys⁵³⁶ to Ala has the ternary form of T7 DNA polymerase resolved the 8-oxoG (syn):dATP basepair 15.

Kinetic data for pol β, RB69, Dpo4, wild-type T7, Lys⁵³⁶Ala T7, and BF 10,11,12,15,16 indicate preference ratios of dCTP over dATP incorporation opposite 8-oxoG to be 2:1, 20:1, ∼70:1, 2:1, 1:20, and 1:9, respectively. We thus can infer that the incorporation of 8-oxoG (syn):dATP mismatch is energetically less favorable in pol β, wild-type T7, RB69, BF, and Dpo4, but more favorable in Lys⁵³⁶Ala T7, than the corresponding 8-oxoG (anti):dCTP basepair.

Although the nascent basepair 8-oxoG:dAMP is distorted in the pol β/DNA crystal structure, a modeling of dATP pairing with syn 8-oxoG in the closed pol β revealed no serious steric clashes at the active site 17. Since pol β maintains fidelity in DNA replication through an induced-fit mechanism—the thumb subdomain of pol β closes when the correct nucleotide complementary to the template residue binds 18,19,20,21,22 and opens after the chemical reaction to release the reaction products and translocate DNA—the induced-fit cycle may also contribute to differentiating dATP and dCTP insertions opposite 8-oxoG and dG12. Specifically, dATP is less efficiently inserted opposite 8-oxoG than dCTP in pol β, though dATP insertion is ∼10⁵ more efficient compared with it opposite dG. Furthermore, computational studies of pol β22,23,24,25,26,27,28 have shown that conformational pathways of pol β before and after the chemical reaction play a vital role in determining its fidelity. With the ultimate goal of understanding the fidelity mechanism of pol β in processing 8-oxoG, we investigate here the dynamic process of pol β's conformational pathways before and after the chemical reaction by performing a series of six dynamics simulations starting from intermediate (partially open) structures where the incoming nucleotides dCTP and dATP pair with 8-oxoG in anti or syn orientations. Although insertion rates of dATP and dCTP opposite 8-oxoG in pol β differ slightly, atomic-level simulations can help unravel systematic differences in their conformational pathways to explain biological observations. In fact, our transition path sampling simulations further dissected the conformational and energetics pathways of correct and incorrect nucleotide insertions opposite 8-oxoG in pol β29. The resolved free energy barriers in that work along with the results here suggest that the different transition states and sequences of conformational events during thumb closing for the two systems could be correlated to different stabilities of the respective closed states and associated insertion efficiencies.

In free duplex DNA, 8-oxoG assumes an anti conformation when pairing with dC and a syn conformation when situated opposite dA30. Besides these two pairing conformations, we also simulated intermediate structures of a pol β/DNA complex with 8-oxoG (anti):dATP to exhaust all possibilities.

The intermediate structures of pol β/DNA complexes were constructed by averaging the PDB entries 1BPX and 1BPY for simulations before chemistry and averaging 1BPY and 1BPZ for simulations after chemistry as in previous works 22,23. We started from these intermediate forms to accelerate any possible motions involved 23; partially open or intermediate pol β structures have been captured in crystal forms 17. The G:C nascent basepairs in the intermediate structures were replaced by 8-oxoG (anti):dC(TP), 8-oxoG (syn):dA(TP), and 8-oxoG (anti):dA(TP) basepairs, respectively. The syn 8-oxoG was obtained by rotating the N-glycosidic bond (N-C1′) of anti 8-oxoG by 180° and relaxing the structure. The missing coordinates for protein residues 1–4 in the open gapped binary complex (1BPX) and 1–9 in the ternary closed complex (1BPY) were modeled using Insight II program. All hydrogen atoms were added using CHARMM 31,32,33. We did not constrain the torsion angle of O3′-P-O5′-C5′ in the simulation for 8-oxoG (anti):dCTP to test whether the steric hindrance between the 8-oxo and O5′ atoms of 8-oxoG drives the template backbone to rotate as observed in the ternary crystal structure 13. Indeed, our simulation did show that this backbone dihedral angle rotates by ∼180° and the O8 ··· O5′ distance increases from 5.7 to 6.8Å.

For simulations before chemistry, the nucleotide-binding and catalytic magnesium ions were kept at the active site. For those after chemistry, the chemical reaction was performed manually by connecting 3′-O of the primer terminus and P_α of the incoming nucleotide. The pyrophosphate product and the binding magnesium ions were removed from the active site.

The six pol β/DNA intermediate complexes (three before and three after chemistry) were solvated in face-centered water cubes using Simulaid 34 and PBCAID 35. The smallest image distance was chosen as 16Å. Water molecules within 1.8Å of the heavy atoms of the crystal structure were removed. To neutralize the system and produce an ionic strength of 150mM, sodium and chloride ions were added by replacing the water oxygens bearing most negative and most positive electrostatic potentials (computed using DELPHI) 36,37, respectively. All ions added were placed at least 8Å away from the protein or DNA or from each other. The final models contain 41,986 atoms for simulations before chemistry (335 protein residues, 32 nucleotides, 2 Mg²⁺ ions, 21 Cl⁻ ions, 43 Na⁺ ions, and 11,830 water molecules), and 41,975 atoms for those after chemistry (335 protein residues, 32 nucleotides, 21 Cl⁻ ions, 44 Na⁺ ions, and 11,830 water molecules).

Periodic boundary conditions and CHARMM27 all-atom force fields for nucleic acids and lipid were used for all energy minimization and MD simulations in CHARMM 32,33.

The force-field parameters for 8-oxoG were adopted from an earlier work 38. The nonbonded interactions were truncated at 14Å, with van der Waal interactions treated by the switch cutoff method and electrostatic interactions treated by atom-based force-shift method. The performance of different long-range truncation methods have been evaluated by Norberg et al. 39 using a highly charged oligonucleotide in aqueous solution. Results show that the atom-based force-shift method can produce very accurate and stable simulation trajectories and that it is computationally more efficient than the particle-mesh Ewald truncation method.

We minimized and equilibrated the models as follows: the water molecules and hydrogen atoms were minimized using the steepest-decent (SD) algorithm for 10,000 steps with all the other heavy atoms fixed, followed by an adopted-basis Newton-Raphson minimization for 20,000 steps; an equilibration of 10ps was performed at 300K by Langevin dynamics to ensure that all the sodium and chloride ions were located on potential energy minima or maxima around the protein/DNA complexes; the entire system was again minimized by SD for 10,000 steps and adopted-basis Newton-Raphson minimization for 20,000 steps with all the protein and DNA heavy atoms fixed; finally, the system was equilibrated with the multiple-timestep Langevin integrator, LN 40,41,42,43, for 100ps with all the atoms released.

For production runs, LN was used with timesteps Δτ/Δt_m/Δt set at 1/2/150 fs for the fast, medium, and slow force components and a medium-range cutoff of 7Å and healing and buffer lengths of 4Å each. The damping constant γ was set to 10ps⁻¹. The structures before chemistry were simulated for ∼18ns and those after chemistry for 13ns. SHAKE was applied to all bonds with hydrogen atoms. Coordinates were saved every 3ps.

Using α-helix N on pol β's thumb subdomain as an index of different enzyme states, we compare the final conformations of simulated complexes to the crystal structures in Fig. 2 (top). Results show that the thumb subdomain closes in the 8-oxoG (anti):dCTP system, while it is partially open in 8-oxoG (syn):dATP and completely open in 8-oxoG (anti):dATP. These trends are also evident from the root mean-square deviation (RMSD) plots of α-helix N in Supplementary Material Fig. S1, a–c . Note that although the models with different nascent basepairs were built from the same intermediate structures (both before and after chemistry), they deviate from the initial coordinates during the minimization/equilibration process so that the starting RMSD values on these plots appear different among the three systems.

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Figure 2

Top and side views of α-helices N in the simulated pol β complexes before (top) and after (bottom) the chemical reaction compared with those in the binary and ternary crystallographic structures of pol β19. Pol β/substrate complexes with the 8-oxoG (anti):dC(TP), 8-oxoG (syn):dA(TP), and 8-oxoG (anti):dA(TP) nascent basepairs are shown in blue, purple, and gray, respectively. Superimposition is performed according to the palm subdomains.

Crystallographic and computational studies 19,22,23,27 have indicated that a sequence of side-chain rearrangements (Asp¹⁹² rotation, Phe²⁷² flip, and Arg²⁵⁸ rotation) occurs at the active site of pol β as it transitions from open to closed states during DNA synthesis. The final conformations of these key residues are displayed in Fig. 3 (top). In the 8-oxoG (anti):dCTP system, Asp¹⁹², Phe²⁷², and Arg²⁵⁸ achieve closed states after the simulation, though Phe²⁷² oscillates between open and closed states. In the two 8-oxoG:dATP complexes, Asp¹⁹² is in a closed conformation binding the two magnesium ions, Arg²⁵⁸ remains at the intermediate state, and Phe²⁷² rotates to the open state (see Supplementary Material Fig. S2 for the time evolutions of dihedral angles).

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Figure 3

Final conformations of the active-site residues in the simulated complexes before (top) and after (bottom) the chemical reaction compared with those in the binary gapped and ternary crystallographic structures. Superimposition is done according to the palm subdomains. The residue side chains and protein backbones in the complexes with 8-oxoG (anti):dC(TP), 8-oxoG (syn):dA(TP), and 8-oxoG (anti):dA(TP) are colored blue, purple, and gray, respectively. The magnesium ions are shown by green spheres.

In sum, thumb and active-site residue motions indicate that pol β closes after the simulation when 8-oxoG (anti):dCTP binds, whereas the complex remains partially and completely open when dATP is opposite syn and anti 8-oxoG, respectively.

Besides these domain and side-chain motions, distortions of the nascent basepairs occur in each 8-oxoG complex. In the complex of syn 8-oxoG pairing with dATP, the N-glycosidic angle of the dATP changes from anti to syn (Figure 4a, left), suggesting unfavorable interactions for dATP incorporation. In contrast, 8-oxoG (anti):dATP and 8-oxoG (anti):dCTP are paired and coplanar (Figure 4bc, left), despite being tilted.

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Figure 4

The final conformations of the nascent basepairs in the simulated complexes before and after the chemical reaction compared with those of the ternary (red) and binary nicked (green) crystallographic structures. Color schemes for the three complexes are as labeled.

More important, even though the thumb and critical residues have attained the closed conformation in the 8-oxoG (anti):dCTP system, a close examination of the active site reveals that the coordination of the catalytic and nucleotidyl-binding magnesium ions is far from the ideal “two-metal-ion catalysis” geometry depicted in Supplementary Material Fig. S3 44,45. Specifically, the catalytic Mg²⁺ does not coordinate with Asp²⁵⁶ but binds 3′-O of the primer terminus, and the P_α ⋯ 3′-O⁻ distance is 6.0Å, much greater than the ideal value of 3.3Å 46. In comparison to the geometry of pol β complexed with G:dCTP 22, the 8-oxoG:dCTP system exhibits large deviations, especially in the binding of the two metal ions, which are positioned farther away from the reaction-competent state. Hence, additional rearrangements are needed to adjust the active-site contents for the nucleotidyl-transfer reaction to occur after the thumb closing (see remarks on the pre-chemistry avenue in Discussion).

Since syn 8-oxoG prefers to pair with dATP by forming a Hoogsteen basepair in DNA duplexes, it is noteworthy that the nascent bases are distorted and pol β does not close completely in this system before chemistry. To identify the cause, we study the conformations of other active-site residues proximal to the nascent basepair.

We note that Tyr²⁷¹ interacts unfavorably with both 8-oxoG and dATP bases. As shown in Figure 5a, unfavorable interactions between Tyr²⁷¹ and the 8-oxoG (syn):dATP basepair in the first 5ns trigger dATP to rotate from anti to syn (Figure 6a) and push 8-oxoG toward the DNA major groove (Figure 6b). After the adenine flip, the interaction energy between 8-oxoG and dATP decreases to nearly zero (Supplementary Material Fig. S4 a ). Furthermore, this base flip also appears to correlate with the thumb subdomain's opening, indicating that distortions caused by Tyr²⁷¹ might hinder the thumb's closing for 8-oxoG (syn):dATP. In comparison, the interactions involving Tyr²⁷¹ in the two complexes with anti 8-oxoG are more favorable (Figure 6bc).

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Figure 5

Interaction energies between the nascent basepair and Tyr²⁷¹ in the simulations before chemistry. Red and blue lines represent the total interaction energies between Tyr²⁷¹ and dATP or dCTP, and between Tyr²⁷¹ and the template 8-oxoG, respectively.

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Figure 6

(a) Time evolution of the N-glycosidic bond torsion angle of dATP. (b) The initial (atoms in cyan, blue, red, and orange are C, N, O, and P, respectively) and final (purple) conformations of the nascent basepair in the 8-oxoG (syn):dATP pol β complex. Superimposition is performed according to the palm subdomain.

In the crystal structures of closed pol β/DNA complexes with G:dCTP and 8-oxoG:dCTP 13,19, Tyr²⁷¹ forms van der Waals contacts with both the sugar ring and base of the incoming nucleotide. The closest atom-to-atom distances are 3.86Å for sugar ring ⋯ Tyr²⁷¹ and 3.23Å for base ⋯ Tyr²⁷¹. In our simulation for 8-oxoG (syn):dATP, it is the six-membered ring of adenine pointing to Tyr²⁷¹ that causes repulsion and triggers dATP's base flip; after the adenine rearrangement, the interactions between dATP and Tyr²⁷¹ become favorable.

Thus, our simulations suggest that when pol β encounters 8-oxoG, Tyr²⁷¹ might deter the dATP incorporation by destabilizing the nascent basepair and hindering the thumb's closing. This role was confirmed by an MD simulation of the Tyr²⁷¹Ala mutant for the mismatch complex, which reveals that the thumb closes quickly from the intermediate state (data not shown).

In the simulations after chemistry, the thumb of pol β opens completely in the two 8-oxoG:dA systems, whereas it closes in 8-oxoG (anti):dC, as shown in Fig. 2 (bottom) (RMSD plots in Supplementary Material Fig. S1, d–f ).

Consistent with corresponding thumb motions, active-site residues Asp¹⁹², Arg²⁵⁸, and Phe²⁷² change to closed conformation in the 8-oxoG (anti):dC system but flip to their open states in 8-oxoG (anti):dA. In contrast, the 8-oxoG (syn):dA system still has Arg²⁵⁸ at the intermediate state after Asp¹⁹² and Phe²⁷² rotate to the open conformation.

Thus, pol β tends to close when anti 8-oxoG pairs with dC but open when syn or anti 8-oxoG pairs with dA. The slow opening of the thumb subdomain for 8-oxoG:dC suggests that it prefers the closed to the open form.

The new basepairs 8-oxoG (syn):dA and 8-oxoG (anti):dC assume Hoogsteen and Watson-Crick conformation after the simulation, respectively (Figure 4ab, right). However, in the 8-oxoG (anti):dA system, dA stacks rather than pairs with the template (Figure 4c, right). The base stacking observed here resembles the A:A mismatch found in the recently solved pol β binary crystal structure 47. The highly distorted 8-oxoG (anti):dA system likely affects the extension rate and induces DNA dissociation and proofreading by an extrinsic exonuclease.

Computational and experimental studies on pol β and other polymerases 38,48,49,50 have suggested that subtle active-site rearrangements may be a key step in the catalytic cycle of these DNA polymerases. In prior works 38,48,51, we have termed these subtle motions in pol β and Dpo4 as the pre-chemistry avenue and emphasized its importance in the overall pathway. Although the conformational closing of pol β before chemistry appears unaffected by the 8-oxo group compared to that in the G:C system 22, the active-site geometry in the former 8-oxoG system deviates further from the reaction-competent state than does the latter complex. Therefore, the energy barriers associated with the pre-chemistry avenue may be larger for the lesioned complex; this difference in energy might contribute to the lower efficiency of dCTP incorporation opposite 8-oxoG than opposite dG in pol β12 (efficiency ratio of 1:4).

In addition, we find that Tyr²⁷¹ competes for the incoming nucleotide site when dATP pairs with a syn 8-oxoG; the unfavorable interactions result in large distortions in the active site destabilizing the closed state. Hence, Tyr²⁷¹ might cause the slow thumb closing and lower insertion efficiency of dATP than dCTP opposite 8-oxoG. A site-directed mutagenesis experiment on Tyr²⁷¹ (e.g., Tyr²⁷¹Ala) might help verify the function of Tyr²⁷¹ in pol β's selection of dCTP against dATP. In fact, a recent mutagenesis experiment has found that mutating Lys⁵³⁶ of T7 DNA polymerase to alanine switches the dCTP/dATP preference 15; Lys⁵³⁶ is a recognized residue that interacts with the templating base.

Structural and kinetic data for correct and incorrect dNTP incorporation in various DNA polymerases show that details of the overall reaction profile of these enzymes and the rate-limiting step are case-dependent (see 52 and therein). For pol β, in particular, experimental measurements using fluorescence-based techniques could not determine exclusively which step is rate-limiting 53,54. Prior dynamics simulations 22,23,24,27,28,51 suggest that the conformational changes before the chemical reaction are rapid and not likely rate-limiting. Our QM/MM studies of the correct (dCTP) and incorrect (dATP) incorporations opposite G51,55 indicate that the chemical step is rate-limiting for both cases. Particularly, pol β's fidelity discrimination is achieved by destabilizing the closed state and increasing the free energy barrier for the deprotonation of 3′-OH when dATP is incorporated opposite G. Since the thumb closing/opening motions in the 8-oxoG pol β complexes occur over nanosecond range, the subtle rearrangements for the divalent metal ions as well as the following nucleotidyl-transfer reaction might be rate-limiting in these complexes. Yet all conformational steps and subtle residue motions that lead to the closed conformation of pol β can affect the reaction efficiency and fidelity.

The partially open form of pol β in the simulated 8-oxoG (syn):dATP system suggests higher free energy barriers for thumb closing. This might increase the dissociation rate of dATP from the active site and lower the binding affinity and incorporation efficiency of dATP opposite 8-oxoG. Further, our simulations also reveal that pol β achieves a more closed conformation when a syn rather than anti 8-oxoG unit pairs with dATP. Because a G:dATP mismatch would resemble the 8-oxoG (anti):dATP pair, a more solvent-exposed active site in a G:dATP system would explain the low insertion efficiencies of dATP opposite G compared to 8-oxoG12.

Taken together, our series of dynamics simulations comparing incorporation of dATP versus dCTP opposite 8-oxoG as well as dCTP opposite the lesioned versus nonlesioned template guanine suggest how pol β's closing and opening conformational pathways affect its fidelity mechanism when processing 8-oxoG. A separate transition path sampling work 29 provides further insights regarding the preference ratio (2:1 from 12). The delineated pathways of the thumb closing for the two 8-oxoG complexes suggest that distinct sequences and energy barriers associated with the conformational events in the 8-oxoG complexes lead to closed forms with different stability and thus different insertion efficiencies 29.

Of course, the MD simulations are never long enough in relation to pol β's function in vivo, so the captured thumb motions only suggest trends (i.e., the preference of the open and closed states of thumb). Force-field imperfections and modeling approximations also apply to all dynamics simulations. Nevertheless, our trajectories clearly distinguish conformational profiles when pol β inserts different incoming nucleotides (dATP or dCTP) opposite the 8-oxoG, and also help interpret existing crystal structures of pol β13,17 with mismatches. The general question of whether mismatched systems actually attain closed state or whether the chemical reaction can proceed from a partially open or open state is an important challenge for future investigations. This question likely corresponds to our notion of the pre-chemistry avenue 51 in which high barriers that are basepair- and system-dependent must be overcome to reach the chemical-reaction competent state.