title: 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-1for 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 pKaof the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.
authors.affiliation: 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.
authors.forename: Martin
authors.identifier: 0000-0002-5076-4936
authors.initials: M
authors.lastname: Klvaňa
authors.affiliation: Laboratory of Physical Chemistry and Chemical Thermodynamics, Faculty of Chemistry and Chemical Technology, University of Maribor , Smetanova ulica 17, 2000 Maribor, Slovenia.|Laboratory for Molecular Modeling, National Institute of Chemistry , Hajdrihova ulica 19, 1001 Ljubljana, Slovenia.
authors.forename: Urban
authors.initials: U
authors.lastname: Bren
authors.affiliation: Department of Chemistry and Biochemistry, Loyola University Chicago , 1032 W. Sheridan Road, Chicago, Illinois 60660, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Florián
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D011522:Protons; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta