Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity

Abstract

The fidelity of DNA polymerases (Pols) refers to their ability to incorporate the correct nucleotide into the growing strand during DNA synthesis. Each Pol operates with a certain degree of fidelity, from high (∼10–8; ∼1 error in 108 bases) to low (∼10–1; ∼1 error in 10 bases) values. The mechanistic factors behind these differences in fidelity are poorly understood. Here, we show that the formation of the Michaelis–Menten complex is critically affected by the metal-mediated dynamics of local structural features at the catalytic center of Pols. We demonstrated this by integrating recent structural and kinetics data of high-fidelity Pol β and low-fidelity Pol η with equilibrium molecular dynamics and free-energy simulations of paired and mispaired reactant complexes of these Pols. We found that local dynamics at the reaction center determines whether the nucleophile is optimally aligned to incorporate the correct (dCTP) or incorrect (dATP) nucleotide opposite a template deoxyguanosine (dG). In Pol β, local structural distortions at the catalytic site are visible only in the dG:dATP mispair complex, which energetically disfavors incorrect nucleotide addition and thus promotes high fidelity. In contrast, in Pol η we observed a more flexible base pair shape complementarity at the catalytic site. This allows reactive configurations of matched and mismatched complexes to be formed with similar ease, thus explaining the low fidelity of Pol η in line with the experimental evidence. Comparisons with other Pols suggest that these local metal-mediated structural dynamics at the reaction center of the catalytic site are crucial to modulating Pol fidelity.

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study

“…Our choice follows the guidelines on site-specific structural features for Mg 2+ ions in RdRp:RNA, as suggested by Casalino et al 47 and in agreement with other computational polymerase studies. 48 , 49 The system was solvated in a orthorhombic box (12 Å from the protein) of TIP3P water molecules. 50 …”

“…Our choice follows the guidelines on site-specific structural features for Mg 2+ ions in RdRp:RNA, as suggested by Casalino et al 47 and in agreement with other computational polymerase studies. 48,49 The system was solvated in a orthorhombic box (12 Å from the protein) of TIP3P water molecules. 50 The solvated structures were first minimized by applying harmonic restraints on all atoms of the enzyme (50 kcal/mol Å 2 ), using 5000 steps of the steepest descent algorithm, followed by 5000 steps of the conjugate gradient algorithm.…”

Alchemical Free-Energy Calculations of Watson–Crick and Hoogsteen Base Pairing Interconversion in DNA

“…System 1 was neutralized by adding Na + ions, and additional Na + and Cl – ions were added to achieve an ionic concentration of 25 mM NaCl, as in the experimental study of Al-Hashimi et al For system 2, K + ions were used instead of Na + for neutralization, and additional Mg 2+ , K + , and Cl – ions were added to achieve ionic concentrations of 50 mM KCl and 2.5 mM MgCl 2 . The protein and DNA were described using the AMBER14ffSB and parmbsc1 force fields, respectively, which we have previously used to study the mechanisms of nucleic-acid-processing enzymes. , The charges for 8OG (Table S1 in the Supporting Information) were derived by multiconformational restrained electrostatic potential fitting, , as explained in more detail in the Supporting Information.…”

“…The protein and DNA were described using the AMBER14ffSB 33 and parmbsc1 27 force fields, respectively, which we have previously used to study the mechanisms of nucleic-acid-processing enzymes. 34 , 35 The charges for 8OG ( Table S1 in the Supporting Information) were derived by multiconformational restrained electrostatic potential fitting, 36 , 37 as explained in more detail in the Supporting Information .…”

TAB1 binding induced p38α conformation change: an accelerated molecular dynamics simulation study

“…Simulation of each system was performed using AMBER18 software [35][36][37] utilizing the ff14SB parameters for proteins. 23 In addition, parameters for the ATP and Mg 2+ ions were taken from Meagher et al 38 and Olof et al, 39 which have been widely used to simulate Mg 2+ -phosphate binding in molecular systems [40][41][42][43][44][45][46] including ATP-Mg 2+ binding in protein kinases. [40][41][42][43][44] All systems were solvated in a cubic box with a minimum distance of 12.0 Å between any system atom and box wall, and 7 Na + ions were added to the solution to neutralize the charge.…”