Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

Abstract

Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3′-OH group of the primer DNA strand, and the subsequent formation and cleavage of P–O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol–1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P–O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3′-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

The structural model of Zika virus RNA-dependent RNA polymerase in complex with RNA for rational design of novel nucleotide inhibitors

“…The catalytic Mg 2+ ion (A) clearly provides the principal linkage of the nascent RNA with the accessing NTP that is activated only upon O3′-deprotonation of the terminal nucleotide within the nascent RNA. This assumption is coherent with recent benchmark calculations of the P-O bond formation operated by DNA Polymerases β and λ 61 . Our QM/MM calculations furthermore revealed that the interactions between the NTP phosphate and other RNA molecules remained preserved, only respective bond distances varied upon altered RNA sequence (Table S5 ).…”

“…The O3′-Pα calculated with Mn 2+ for normal and O3′-deprotonated ribose of nucleotide 8 was 3.268 Å and 3.076 Å, respectively. The activated state of the RNA substrate, however, assumes O3′-deprotonation of the terminal nucleoside within the nascent RNA that takes place in the catalytic core 61 . Consequently, the majority of the QM/MM calculations employed O3′-deprotonated nucleotide 8 to illustrate the reaction state shortly before formation of the O3′-Pα phosphodiester bond.…”

Molecular Mechanisms of DNA Replication and Repair Machinery: Insights from Microscopic Simulations

“…One can attempt to compute the free energy difference between the states with either the exact or approximate expression using either Monte Carlo or MD to sample molecular configurations . The FEP method has been used to study the fidelity of DNA replication by computing the free‐energy of mismatched structures in complex with protein and for evaluation of repair pathways …”

“…[]. The EVB method was used to study the synthesis of DNA during replication and base excision repair of DNA damage . The EVB method is much less computationally demanding than the conventional ab initio QM/MM method and is able to faithfully describe catalytic effects and enzyme–solvent interactions.…”

“…Pol β is the best computationally studied polymerase . Most studies have explored the energetics and possible pathways of the reaction for intact basepairs . A few studies have explored the relationship between large‐scale conformation of the repair polymerase and its fidelity .…”