An abridged transition state model to derive structure, dynamics, and energy components of DNA polymerase β fidelity.

DNA base-excision repair involving human DNA polymerase β (pol β) is an essential defense mechanism against genetic alterations that may typically result in cancer in humans (1-4). Biochemical (5) and crystallographic (6) studies, including a recent pol β-DNA-dNTP ternary complexes with incorrect dNTP substrates (7) provide insight in determining the role of conformational and structural factors involved in pol β mechanisms and fidelity (1). It has been suggested that the chemical step is rate-limiting in pol β–catalyzed nucleotide insertion based on studies of time-resolved fluorescence in conjunction with rhodium(III) metals and solvent viscosity alterations (8-10), and by the observation of Bronsted linear free-energy relationships (LFER) for dNTP substrates with β–γ bridging oxygen replaced by haloalkanes (11, 12). Phosphodiester bond hydrolysis in aqueous solution is associated with a broad high-energy transition state region, which allows either distance between the alpha phosphorus of the incoming dNTP and O3’ of the primer nucleotide (Pα-Onuc) or between Pα and leaving group oxygen of the dNTP (Olg) to be between 1.7 to 2.4 A, provided that the sum of these distances is less than 4.0 A (13, 14). All these substrate geometries need to be stabilized in the active site of enzymes that catalyze this class of reaction, DNA polymerase being a prominent example. Thus, a rigorous study of the protein or substrate modifications on enzyme catalysis requires an accurate evaluation of the complete reaction free energy surface for each protein or substrate variant - a task that is currently far too demanding to be practically useful. However, it has been proposed that it may be possible to effectively reduce this surface substantially by selecting a suitable subset of geometries (15). Theoretical computational analyses of transition states (TS) for polymerase-catalyzed deoxyribonucleotide insertion have employed several variants of quantum mechanics/molecular mechanics (QM/MM) (11, 13, 16-21). For the chemical reaction in pol β, an ONIOM (a specific type of QM/MM calculation) analysis suggests that the highest activation barrier occurs early on the nucleotidyl transfer reaction coordinate (17, 22). This early transition state featured a longer Pα–Onuc bond than Pα–Olg bond. Subsequent empirical valence bond (EVB) calculations suggested a late semi-concerted TS to be rate-limiting, but the free energies of early TS configurations were only marginally lower, thus retaining the early TS as a plausible option for the rate-limiting TS (11, 12, 23). Owing, however, to limited sampling of the TS region of the free energy surface during ONIOM or EVB calculations, dynamical aspects of the active site elements stabilizing the TS were not determined. A far more efficient sampling of the TS region uses molecular dynamics (MD) simulations on the TS model generated using classical MM force fields (24, 25). This type of sampling was applied in recent studies of TS dynamics in RNAse A (26), and to the effects of amino acid substitutions on pol β fidelity (15, 27). To address enzyme catalysis, it is necessary to compare the simulated TS properties either to the same TS in aqueous solution (15, 27), or to a ground-state of the enzymatic reaction (26). However, because the 3’-terminal deoxyribose of the primer strand carries an extra proton in the reactant state, the primer-dNTP substrate carries a different total charge in the ground- and transition-states of the nucleotidyl transfer reaction. This variation in the overall charge significantly diminishes the utility of computer simulations to provide consistent energetic and structural comparisons. To avoid the inaccuracies inherent in the computational treatment of long-range electrostatic interactions in biomolecular simulations (28), here we employ a ground state model that is activated by deprotonating Onuc (GSA) as the initial state in an electrostatic linear free energy response (EFER) model. We further postulate a TS structure that geometrically mimics the TS for the nucleophilic attack, but wherein its atomic charges correspond to a fully developed dianionic pentavalent phosphorane intermediate (PTS). The MD simulations on GSA and PTS states are then used to monitor differential interactions of the charges of the probe region of the GSA and PTS states with their respective active site or aqueous solution surroundings. The probe region of the substrate is designed to have its total charge close to zero by including α- and β-phosphates of dNTP, Onuc, and the catalytic Mg2+ ion. The proposed linear-response treatment is reminiscent of the model used in our earlier simulations of relative dNTP binding, in which the probe region represents atoms of the dNTP base in charged and uncharged states, and the DNA-pol β complex with the right or wrong templating bases represents the probe surroundings (29). The structural aspects of catalysis are described by comparing the dynamics of the contacts of pol β active site residues with the primer-dNTP substrate in the GSA and PTS states. Such a structural analysis effectively extends the reach of X-ray diffraction analysis to elusive short-lived species. Classical MD simulations are capable of generating a detailed dynamical picture of any part of the protein internal architecture, often leading to an overabundance of model-dependent structural data. To focus on pol β catalysis, we emphasize those geometric features that differ significantly between simulations of the PTS and GSA complexes. To expand this analysis to pol β fidelity, we compare the same set of geometric features for pol β bound to a model PTS containing either a right or wrong dNTP substrate.