Document Detail


Catalytic effects of mutations of distant protein residues in human DNA polymerase β: theory and experiment.
MedLine Citation:
PMID:  23013478     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol β mutants.
Authors:
Martin Klvaňa; Drew L Murphy; Petr Jeřábek; Myron F Goodman; Arieh Warshel; Joann B Sweasy; Jan Florián
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-10-29
Journal Detail:
Title:  Biochemistry     Volume:  51     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-06     Completed Date:  2013-01-10     Revised Date:  2014-04-13    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  8829-43     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acid Substitution
Base Sequence
Catalysis
DNA Polymerase beta / chemistry*,  genetics*
Deoxyribonucleotides / genetics,  metabolism
Humans
Kinetics
Molecular Dynamics Simulation
Point Mutation
Thermodynamics
Grant Support
ID/Acronym/Agency:
5U19CA105010/CA/NCI NIH HHS; U19 CA105010/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Deoxyribonucleotides; EC 2.7.7.-/DNA Polymerase beta
Comments/Corrections

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