Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite 8-Oxo-7,8-dihydroguanine by Escherichia coli Polymerases I exo- and II exo- †
Identifieur interne : 003E53 ( Main/Exploration ); précédent : 003E52; suivant : 003E54Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite 8-Oxo-7,8-dihydroguanine by Escherichia coli Polymerases I exo- and II exo- †
Auteurs : Laura G. Lowe [États-Unis] ; F. Peter Guengerich [États-Unis]Source :
- Biochemistry [ 0006-2960 ] ; 1996.
Abstract
Escherichia coli polymerases (pol) I exo- (KF-) and pol II exo- (pol II-) were used as model enzymes with a DNA primer/template complex (12/16-mer) to examine the kinetics of incorporation of dCTP and dATP at the site of an 8-oxo-7,8-dihydroguanine (8-oxoGua) residue, compared to guanine (Gua). In steady-state assays (with DNA in excess) the rate of incorporation (kcat) was dCTP > dATP and the Km,dATP < Km,dCTP during incorporation opposite 8-oxoGua with both polymerases. Pre-steady-state kinetic curves (rapid-quench analysis) for the addition of C opposite 8-oxoGua or Gua by KF- and pol II- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of A opposite 8-oxoGua did not display burst kinetics with either enzyme. Reduced rates of incorporation of the dCTPαS and dATPαS phosphorothioate analogs suggest that the rates of incorporation of A and C opposite 8-oxoGua are limited during polymerization by the rate of phosphodiester bond formation. Neither polymerase appears to discriminate between adducted and nonadducted DNA substrate for binding. Kinetic assays performed with varying dCTP concentrations indicate that dCTP has a higher Kd and lower kp (polymerization rate) for incorporation opposite 8-oxoGua compared to Gua. Furthermore, the dATP binding affinities with KF- and pol II- were ∼10- and ∼3-fold lower, respectively, than that of dCTP as determined in competition assays with mixtures of dCTP and dATP. Microscopic rate constants were estimated by mathematical analysis of dNTP concentration dependence curves. Both polymerases preferentially extended the A:8-oxoGua pair while extension of the C:8-oxoGua pair was greatly impaired. Based on these findings, the fidelity of KF- and pol II- during replication of 8-oxoGua depends on contributions from nucleotide binding, the rate of phosphodiester bond formation, and the ease of base pair extension.
Url:
DOI: 10.1021/bi960485x
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Istex, to step Corpus: 001197
- to stream Istex, to step Curation: 001197
- to stream Istex, to step Checkpoint: 001589
- to stream Main, to step Merge: 003F08
- to stream Main, to step Curation: 003E53
Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite 8-Oxo-7,8-dihydroguanine by Escherichia coli Polymerases I exo- and II exo- †</title><author><name sortKey="Lowe, Laura G" sort="Lowe, Laura G" uniqKey="Lowe L" first="Laura G." last="Lowe">Laura G. Lowe</name></author><author><name sortKey="Guengerich, F Peter" sort="Guengerich, F Peter" uniqKey="Guengerich F" first="F. Peter" last="Guengerich">F. Peter Guengerich</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:23F1B3018D329F5FA961FCEB5287914F51C82DD3</idno><date when="1996" year="1996">1996</date><idno type="doi">10.1021/bi960485x</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-Z08XCXLQ-F/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001197</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001197</idno><idno type="wicri:Area/Istex/Curation">001197</idno><idno type="wicri:Area/Istex/Checkpoint">001589</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">001589</idno><idno type="wicri:doubleKey">0006-2960:1996:Lowe L:steady:state:and</idno><idno type="wicri:Area/Main/Merge">003F08</idno><idno type="wicri:Area/Main/Curation">003E53</idno><idno type="wicri:Area/Main/Exploration">003E53</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite
8-Oxo-7,8-dihydroguanine by <hi rend="italic">Escherichia coli</hi> Polymerases I exo<hi rend="superscript">-</hi> and II exo<hi rend="superscript">-</hi>
<ref type="bib" target="#bi960485xAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Lowe, Laura G" sort="Lowe, Laura G" uniqKey="Lowe L" first="Laura G." last="Lowe">Laura G. Lowe</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Tennessee</region></placeName><wicri:cityArea>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville</wicri:cityArea></affiliation></author><author><name sortKey="Guengerich, F Peter" sort="Guengerich, F Peter" uniqKey="Guengerich F" first="F. Peter" last="Guengerich">F. Peter Guengerich</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Tennessee</region></placeName><wicri:cityArea>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville</wicri:cityArea></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1996</date><date type="published">1996</date><biblScope unit="vol">35</biblScope><biblScope unit="issue">30</biblScope><biblScope unit="page" from="9840">9840</biblScope><biblScope unit="page" to="9849">9849</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Escherichia coli polymerases (pol) I exo- (KF-) and pol II exo- (pol II-) were used as model enzymes with a DNA primer/template complex (12/16-mer) to examine the kinetics of incorporation of dCTP and dATP at the site of an 8-oxo-7,8-dihydroguanine (8-oxoGua) residue, compared to guanine (Gua). In steady-state assays (with DNA in excess) the rate of incorporation (kcat) was dCTP > dATP and the Km,dATP < Km,dCTP during incorporation opposite 8-oxoGua with both polymerases. Pre-steady-state kinetic curves (rapid-quench analysis) for the addition of C opposite 8-oxoGua or Gua by KF- and pol II- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of A opposite 8-oxoGua did not display burst kinetics with either enzyme. Reduced rates of incorporation of the dCTPαS and dATPαS phosphorothioate analogs suggest that the rates of incorporation of A and C opposite 8-oxoGua are limited during polymerization by the rate of phosphodiester bond formation. Neither polymerase appears to discriminate between adducted and nonadducted DNA substrate for binding. Kinetic assays performed with varying dCTP concentrations indicate that dCTP has a higher Kd and lower kp (polymerization rate) for incorporation opposite 8-oxoGua compared to Gua. Furthermore, the dATP binding affinities with KF- and pol II- were ∼10- and ∼3-fold lower, respectively, than that of dCTP as determined in competition assays with mixtures of dCTP and dATP. Microscopic rate constants were estimated by mathematical analysis of dNTP concentration dependence curves. Both polymerases preferentially extended the A:8-oxoGua pair while extension of the C:8-oxoGua pair was greatly impaired. Based on these findings, the fidelity of KF- and pol II- during replication of 8-oxoGua depends on contributions from nucleotide binding, the rate of phosphodiester bond formation, and the ease of base pair extension.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Tennessee</li></region></list><tree><country name="États-Unis"><region name="Tennessee"><name sortKey="Lowe, Laura G" sort="Lowe, Laura G" uniqKey="Lowe L" first="Laura G." last="Lowe">Laura G. Lowe</name></region><name sortKey="Guengerich, F Peter" sort="Guengerich, F Peter" uniqKey="Guengerich F" first="F. Peter" last="Guengerich">F. Peter Guengerich</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 003E53 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 003E53 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= ISTEX:23F1B3018D329F5FA961FCEB5287914F51C82DD3
|texte= Steady-State and Pre-Steady-State Kinetic Analysis of dNTP Insertion Opposite 8-Oxo-7,8-dihydroguanine by Escherichia coli Polymerases I exo- and II exo- †
}}
| This area was generated with Dilib version V0.6.33. |  |