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J Phys Chem B. 2016 Dec 29;120(51):13017-13030. doi: 10.1021/acs.jpcb.6b08581. Epub 2016 Dec 19.

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

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Laboratory of Physical Chemistry and Chemical Thermodynamics, Faculty of Chemistry and Chemical Technology, University of Maribor , Smetanova ulica 17, 2000 Maribor, Slovenia.
Department of Chemistry and Biochemistry, Loyola University Chicago , 1032 W. Sheridan Road, Chicago, Illinois 60660, United States.
Laboratory for Molecular Modeling, National Institute of Chemistry , Hajdrihova ulica 19, 1001 Ljubljana, Slovenia.


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.

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