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Statements

Subject Item
n2:RIV%2F00216208%3A11310%2F12%3A10125572%21RIV13-MSM-11310___
rdf:type
skos:Concept n15:Vysledek
rdfs:seeAlso
http://dx.doi.org/10.1021/bi300783t
dcterms:description
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 beta (Pol beta). Since the mutated residues in the point mutants, I174S, 1260Q M282L, H285D, E288K, and K289M, were not located in the Pol beta 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, Omega = (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 beta 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 Omega values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Omega 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 Omega values. 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 beta (Pol beta). Since the mutated residues in the point mutants, I174S, 1260Q M282L, H285D, E288K, and K289M, were not located in the Pol beta 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, Omega = (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 beta 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 Omega values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Omega 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 Omega values.
dcterms:title
Catalytic Effects of Mutations of Distant Protein Residues in Human DNA Polymerase beta: Theory and Experiment Catalytic Effects of Mutations of Distant Protein Residues in Human DNA Polymerase beta: Theory and Experiment
skos:prefLabel
Catalytic Effects of Mutations of Distant Protein Residues in Human DNA Polymerase beta: Theory and Experiment Catalytic Effects of Mutations of Distant Protein Residues in Human DNA Polymerase beta: Theory and Experiment
skos:notation
RIV/00216208:11310/12:10125572!RIV13-MSM-11310___
n15:predkladatel
n16:orjk%3A11310
n3:aktivita
n13:S n13:I
n3:aktivity
I, S
n3:cisloPeriodika
44
n3:dodaniDat
n4:2013
n3:domaciTvurceVysledku
n8:5771943
n3:druhVysledku
n12:J
n3:duvernostUdaju
n17:S
n3:entitaPredkladatele
n20:predkladatel
n3:idSjednocenehoVysledku
126125
n3:idVysledku
RIV/00216208:11310/12:10125572
n3:jazykVysledku
n6:eng
n3:klicovaSlova
force-field; transition-state; molecular-dynamics; replication fidelity; active-site; induced-fit mechanism; amino-acid substitution; free-energy calculations; mispaired primer termini; range electrostatic interactions
n3:klicoveSlovo
n5:force-field n5:transition-state n5:molecular-dynamics n5:active-site n5:range%20electrostatic%20interactions n5:mispaired%20primer%20termini n5:amino-acid%20substitution n5:free-energy%20calculations n5:replication%20fidelity n5:induced-fit%20mechanism
n3:kodStatuVydavatele
US - Spojené státy americké
n3:kontrolniKodProRIV
[CED0CE2D97D5]
n3:nazevZdroje
Biochemistry
n3:obor
n10:CE
n3:pocetDomacichTvurcuVysledku
1
n3:pocetTvurcuVysledku
7
n3:rokUplatneniVysledku
n4:2012
n3:svazekPeriodika
51
n3:tvurceVysledku
Sweasy, Joann B. Goodman, Myron F. Jeřábek, Petr Murphy, Drew L. Warshel, Arieh Florián, Jan Klvaňa, Martin
n3:wos
000310664200013
s:issn
0006-2960
s:numberOfPages
15
n18:doi
10.1021/bi300783t
n19:organizacniJednotka
11310