| Catalytic effects of mutations of distant protein residues in human DNA polymerase β: theory and experiment. | |
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MedLine Citation:
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PMID: 23013478 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Martin Klvaňa; Drew L Murphy; Petr Jeřábek; Myron F Goodman; Arieh Warshel; Joann B Sweasy; Jan Florián |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2012-10-29 |
Journal Detail:
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Title: Biochemistry Volume: 51 ISSN: 1520-4995 ISO Abbreviation: Biochemistry Publication Date: 2012 Nov |
Date Detail:
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Created Date: 2012-11-06 Completed Date: 2013-01-10 Revised Date: 2013-02-28 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 8829-43 Citation Subset: IM |
Affiliation:
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Department of Chemistry, Loyola University, Chicago, Illinois 60626, United States. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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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:
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5U19CA105010/CA/NCI NIH HHS; U19 CA105010/CA/NCI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Deoxyribonucleotides; EC 2.7.7.-/DNA Polymerase beta |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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