MersV1, Istex, Corpus, bibRecord, 002573

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase

Identifieur interne : 002573 ( Istex/Corpus ); précédent : 002572; suivant : 002574

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase

Auteurs : Miguel De Vega ; Luis Blanco ; Margarita Salas

Source :

Journal of Molecular Biology [ 0022-2836 ] ; 1999.

RBID : ISTEX:D2D35156BC771000BCDB79DD32F8B52764484B16

English descriptors

KwdEn :
- DNA polymerase active sites, Pol Ik, TP, dsDNA double-stranded DNA, primer transfer, ssDNA, ø29 DNA polymerase.
Teeft :
- Activator, Active sites, Autoradiography, Bacteriophage, Beese, Benkovic, Bernad, Biol, Blanco, Blanco salas, Bovine serum albumin, Challenger, Chem, Coli, Datp, Delity, Dithiothreitol, Dntp, Dntps, Dsdna, Editing mode, Enzyme surface, Escherichia coli, Exonuclease, Exonuclease activity, Exonucleolysis, Exonucleolytic, Exonucleolytic degradation, Hybrid, Hybrid molecule, Hybridized, Lazaro, Metal activator, Mgcl2, Mutant, Mutational analysis, Nucleotide, Oligo, Oligos, Other hand, Polyacrylamide, Polymerase, Polymerase transfers, Polymerization, Polymerization site, Primer, Primer strand, Primer terminus, Processive, Reaction mixture, Replication, Salas, Sp1biot, Sp1c, Ssdna, Steitz, Strand displacement conditions, Streptavidin, Template strand, Terminus, Truniger, Vega, Wang.

Abstract

Abstract: ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Url:

https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/fulltext.pdf

DOI: 10.1006/jmbi.1999.3052

Links to Exploration step

ISTEX:D2D35156BC771000BCDB79DD32F8B52764484B16

Le document en format XML

<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
<author><name sortKey="De Vega, Miguel" sort="De Vega, Miguel" uniqKey="De Vega M" first="Miguel" last="De Vega">Miguel De Vega</name>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Blanco, Luis" sort="Blanco, Luis" uniqKey="Blanco L" first="Luis" last="Blanco">Luis Blanco</name>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Salas, Margarita" sort="Salas, Margarita" uniqKey="Salas M" first="Margarita" last="Salas">Margarita Salas</name>
<affiliation><mods:affiliation>E-mail: msalas@cbm.uam.es</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">ISTEX</idno>
<idno type="RBID">ISTEX:D2D35156BC771000BCDB79DD32F8B52764484B16</idno>
<date when="1999" year="1999">1999</date>
<idno type="doi">10.1006/jmbi.1999.3052</idno>
<idno type="url">https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/fulltext.pdf</idno>
<idno type="wicri:Area/Istex/Corpus">002573</idno>
<idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002573</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
<author><name sortKey="De Vega, Miguel" sort="De Vega, Miguel" uniqKey="De Vega M" first="Miguel" last="De Vega">Miguel De Vega</name>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Blanco, Luis" sort="Blanco, Luis" uniqKey="Blanco L" first="Luis" last="Blanco">Luis Blanco</name>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
<author><name sortKey="Salas, Margarita" sort="Salas, Margarita" uniqKey="Salas M" first="Margarita" last="Salas">Margarita Salas</name>
<affiliation><mods:affiliation>E-mail: msalas@cbm.uam.es</mods:affiliation>
</affiliation>
<affiliation><mods:affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</mods:affiliation>
</affiliation>
</author>
</analytic>
<monogr></monogr>
<series><title level="j">Journal of Molecular Biology</title>
<title level="j" type="abbrev">YJMBI</title>
<idno type="ISSN">0022-2836</idno>
<imprint><publisher>ELSEVIER</publisher>
<date type="published" when="1999">1999</date>
<biblScope unit="volume">292</biblScope>
<biblScope unit="issue">1</biblScope>
<biblScope unit="page" from="39">39</biblScope>
<biblScope unit="page" to="51">51</biblScope>
</imprint>
<idno type="ISSN">0022-2836</idno>
</series>
</biblStruct>
</sourceDesc>
<seriesStmt><idno type="ISSN">0022-2836</idno>
</seriesStmt>
</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>DNA polymerase active sites</term>
<term>Pol Ik</term>
<term>TP</term>
<term>dsDNA double-stranded DNA</term>
<term>primer transfer</term>
<term>ssDNA</term>
<term>ø29 DNA polymerase</term>
</keywords>
<keywords scheme="Teeft" xml:lang="en"><term>Activator</term>
<term>Active sites</term>
<term>Autoradiography</term>
<term>Bacteriophage</term>
<term>Beese</term>
<term>Benkovic</term>
<term>Bernad</term>
<term>Biol</term>
<term>Blanco</term>
<term>Blanco salas</term>
<term>Bovine serum albumin</term>
<term>Challenger</term>
<term>Chem</term>
<term>Coli</term>
<term>Datp</term>
<term>Delity</term>
<term>Dithiothreitol</term>
<term>Dntp</term>
<term>Dntps</term>
<term>Dsdna</term>
<term>Editing mode</term>
<term>Enzyme surface</term>
<term>Escherichia coli</term>
<term>Exonuclease</term>
<term>Exonuclease activity</term>
<term>Exonucleolysis</term>
<term>Exonucleolytic</term>
<term>Exonucleolytic degradation</term>
<term>Hybrid</term>
<term>Hybrid molecule</term>
<term>Hybridized</term>
<term>Lazaro</term>
<term>Metal activator</term>
<term>Mgcl2</term>
<term>Mutant</term>
<term>Mutational analysis</term>
<term>Nucleotide</term>
<term>Oligo</term>
<term>Oligos</term>
<term>Other hand</term>
<term>Polyacrylamide</term>
<term>Polymerase</term>
<term>Polymerase transfers</term>
<term>Polymerization</term>
<term>Polymerization site</term>
<term>Primer</term>
<term>Primer strand</term>
<term>Primer terminus</term>
<term>Processive</term>
<term>Reaction mixture</term>
<term>Replication</term>
<term>Salas</term>
<term>Sp1biot</term>
<term>Sp1c</term>
<term>Ssdna</term>
<term>Steitz</term>
<term>Strand displacement conditions</term>
<term>Streptavidin</term>
<term>Template strand</term>
<term>Terminus</term>
<term>Truniger</term>
<term>Vega</term>
<term>Wang</term>
</keywords>
</textClass>
<langUsage><language ident="en">en</language>
</langUsage>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">Abstract: ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.</div>
</front>
</TEI>
<istex><corpusName>elsevier</corpusName>
<keywords><teeft><json:string>polymerase</json:string>
<json:string>primer</json:string>
<json:string>exonuclease</json:string>
<json:string>blanco</json:string>
<json:string>primer strand</json:string>
<json:string>biol</json:string>
<json:string>salas</json:string>
<json:string>streptavidin</json:string>
<json:string>sp1c</json:string>
<json:string>chem</json:string>
<json:string>mutant</json:string>
<json:string>primer terminus</json:string>
<json:string>exonucleolytic</json:string>
<json:string>lazaro</json:string>
<json:string>processive</json:string>
<json:string>oligo</json:string>
<json:string>datp</json:string>
<json:string>sp1biot</json:string>
<json:string>polyacrylamide</json:string>
<json:string>template strand</json:string>
<json:string>ssdna</json:string>
<json:string>metal activator</json:string>
<json:string>mgcl2</json:string>
<json:string>activator</json:string>
<json:string>dntps</json:string>
<json:string>wang</json:string>
<json:string>bernad</json:string>
<json:string>autoradiography</json:string>
<json:string>replication</json:string>
<json:string>terminus</json:string>
<json:string>steitz</json:string>
<json:string>coli</json:string>
<json:string>bacteriophage</json:string>
<json:string>benkovic</json:string>
<json:string>nucleotide</json:string>
<json:string>hybrid</json:string>
<json:string>dsdna</json:string>
<json:string>dithiothreitol</json:string>
<json:string>dntp</json:string>
<json:string>truniger</json:string>
<json:string>other hand</json:string>
<json:string>oligos</json:string>
<json:string>bovine serum albumin</json:string>
<json:string>reaction mixture</json:string>
<json:string>beese</json:string>
<json:string>vega</json:string>
<json:string>delity</json:string>
<json:string>exonuclease activity</json:string>
<json:string>exonucleolysis</json:string>
<json:string>hybridized</json:string>
<json:string>exonucleolytic degradation</json:string>
<json:string>polymerization site</json:string>
<json:string>active sites</json:string>
<json:string>escherichia coli</json:string>
<json:string>challenger</json:string>
<json:string>enzyme surface</json:string>
<json:string>editing mode</json:string>
<json:string>hybrid molecule</json:string>
<json:string>blanco salas</json:string>
<json:string>mutational analysis</json:string>
<json:string>strand displacement conditions</json:string>
<json:string>polymerase transfers</json:string>
<json:string>polymerization</json:string>
<json:string>degradation</json:string>
<json:string>strand</json:string>
<json:string>polymerization mode</json:string>
<json:string>gopalakrishnan benkovic</json:string>
<json:string>exonucleolytic removal</json:string>
<json:string>exonuclease site</json:string>
<json:string>polymerization errors</json:string>
<json:string>elongation products</json:string>
<json:string>loading buffer</json:string>
<json:string>crystal structure</json:string>
<json:string>assay</json:string>
</teeft>
</keywords>
<author><json:item><name>Miguel de Vega</name>
<affiliations><json:string>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</json:string>
</affiliations>
</json:item>
<json:item><name>Luis Blanco</name>
<affiliations><json:string>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</json:string>
</affiliations>
</json:item>
<json:item><name>Margarita Salas</name>
<affiliations><json:string>E-mail: msalas@cbm.uam.es</json:string>
<json:string>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</json:string>
</affiliations>
</json:item>
</author>
<subject><json:item><lang><json:string>eng</json:string>
</lang>
<value>Regular article</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>ø29 DNA polymerase</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>primer transfer</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>DNA polymerase active sites</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>Pol Ik : Klenow fragment of DNA polymerase I from E.coli</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>ssDNA : single-stranded DNA</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>dsDNA double-stranded DNA</value>
</json:item>
<json:item><lang><json:string>eng</json:string>
</lang>
<value>TP : terminal protein</value>
</json:item>
</subject>
<arkIstex>ark:/67375/6H6-NC1962KS-H</arkIstex>
<language><json:string>eng</json:string>
</language>
<originalGenre><json:string>Full-length article</json:string>
</originalGenre>
<abstract>Abstract: ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.</abstract>
<qualityIndicators><score>8.836</score>
<pdfWordCount>8738</pdfWordCount>
<pdfCharCount>51494</pdfCharCount>
<pdfVersion>1.4</pdfVersion>
<pdfPageCount>13</pdfPageCount>
<pdfPageSize>612 x 792 pts (letter)</pdfPageSize>
<refBibsNative>true</refBibsNative>
<abstractWordCount>153</abstractWordCount>
<abstractCharCount>1031</abstractCharCount>
<keywordCount>8</keywordCount>
</qualityIndicators>
<title>Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
<pmid><json:string>10493855</json:string>
</pmid>
<pii><json:string>S0022-2836(99)93052-8</json:string>
</pii>
<genre><json:string>research-article</json:string>
</genre>
<host><title>Journal of Molecular Biology</title>
<language><json:string>unknown</json:string>
</language>
<publicationDate>1999</publicationDate>
<issn><json:string>0022-2836</json:string>
</issn>
<pii><json:string>S0022-2836(00)X0290-2</json:string>
</pii>
<volume>292</volume>
<issue>1</issue>
<pages><first>39</first>
<last>51</last>
</pages>
<genre><json:string>journal</json:string>
</genre>
</host>
<namedEntities><unitex><date><json:string>1999</json:string>
</date>
<geogName></geogName>
<orgName><json:string>Biotech</json:string>
<json:string>Operon Technologies</json:string>
<json:string>Fundacion Ramon Areces</json:string>
</orgName>
<orgName_funder><json:string>Fundacion Ramon Areces</json:string>
</orgName_funder>
<orgName_provider></orgName_provider>
<persName><json:string>A. Bonnin</json:string>
<json:string>Margarita Salas</json:string>
<json:string>Pol Ik</json:string>
<json:string>L. Blanco</json:string>
<json:string>Luis Blanco</json:string>
<json:string>Miguel de Vega</json:string>
<json:string>M. Salas</json:string>
<json:string>Eladio Vinuela</json:string>
<json:string>J.M. Lazaro</json:string>
</persName>
<placeName><json:string>Mannheim</json:string>
<json:string>European Union</json:string>
<json:string>Spain</json:string>
</placeName>
<ref_url><json:string>http://www.idealibrary.com</json:string>
</ref_url>
<ref_bibl><json:string>Weisshart et al., 1994</json:string>
<json:string>Truniger et al., 1996</json:string>
<json:string>reviewed by Blanco & Salas, 1996</json:string>
<json:string>Ândez et al., 1992</json:string>
<json:string>Das & Fujimura, 1985</json:string>
<json:string>Beese & Steitz, 1991</json:string>
<json:string>Joyce & Steitz, 1987</json:string>
<json:string>Mendez et al., 1994</json:string>
<json:string>Derbyshire et al., 1995</json:string>
<json:string>Pisani & Rossi, 1994</json:string>
<json:string>Kunkel, 1992</json:string>
<json:string>Joyce, 1989</json:string>
<json:string>Donlin et al., 1991</json:string>
<json:string>intermolecular Pol-Exo transference; Joyce, 1989</json:string>
<json:string>Me Truniger et al., 1996</json:string>
<json:string>Esteban et al., 1994</json:string>
<json:string>Saturno et al., 1997, 1998</json:string>
<json:string>Bernad et al., 1990</json:string>
<json:string>Wang et al., 1997</json:string>
<json:string>Blanco & Salas, 1995, 1996</json:string>
<json:string>Capson et al., 1991</json:string>
<json:string>Donlin et al. (1991)</json:string>
<json:string>Blanco et al., 1989</json:string>
<json:string>Allen et al., 1989</json:string>
<json:string>Studier & Moffatt, 1986</json:string>
<json:string>Bernad et al., 1989</json:string>
<json:string>Olson & Kaguni, 1992</json:string>
<json:string>Yang & Richardson, 1996</json:string>
<json:string>Guest et al., 1991</json:string>
<json:string>Stocky et al., 1995</json:string>
<json:string>Wang et al., 1996</json:string>
<json:string>Gopalakrishnan & Benkovic, 1994</json:string>
<json:string>de Vega et al., 1996, 1998a,b</json:string>
<json:string>de Vega et al., 1996, 1997, 1998a,b</json:string>
<json:string>Truniger et al., 1998</json:string>
<json:string>Eom et al., 1996</json:string>
<json:string>Beese et al., 1993</json:string>
<json:string>Beese et al.</json:string>
<json:string>Blasco et al., 1995</json:string>
<json:string>Catalano et al., 1990</json:string>
<json:string>Ollis et al., 1985</json:string>
<json:string>Strick & Knopf, 1998</json:string>
<json:string>reviewed by Blanco & Salas, 1995, 1996</json:string>
<json:string>Garmendia et al., 1992</json:string>
<json:string>Kiefer et al., 1997,1998</json:string>
<json:string>Doublie et al., 1998</json:string>
<json:string>Blanco & Salas, 1995</json:string>
<json:string>King et al., 1997</json:string>
<json:string>Weber et al., 1989</json:string>
<json:string>Derbyshire et al., 1991</json:string>
<json:string>Reddy et al., 1992</json:string>
<json:string>Lin et al., 1994</json:string>
<json:string>Munn & Alberts, 1991</json:string>
<json:string>Baker & Reha-Krantz, 1998</json:string>
<json:string>4-5 nt to the exo site and 7 nt to the pol site; Gopalakrishnan & Benkovic, 1994</json:string>
<json:string>Lazaro et al., 1995</json:string>
</ref_bibl>
<bibl></bibl>
</unitex>
</namedEntities>
<ark><json:string>ark:/67375/6H6-NC1962KS-H</json:string>
</ark>
<categories><wos><json:string>1 - science</json:string>
<json:string>2 - biochemistry & molecular biology</json:string>
</wos>
<scienceMetrix><json:string>1 - health sciences</json:string>
<json:string>2 - biomedical research</json:string>
<json:string>3 - biochemistry & molecular biology</json:string>
</scienceMetrix>
<scopus><json:string>1 - Life Sciences</json:string>
<json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string>
<json:string>3 - Molecular Biology</json:string>
</scopus>
<inist><json:string>1 - sciences appliquees, technologies et medecines</json:string>
<json:string>2 - sciences biologiques et medicales</json:string>
<json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string>
<json:string>4 - biologie moleculaire et cellulaire</json:string>
</inist>
</categories>
<publicationDate>1999</publicationDate>
<copyrightDate>1999</copyrightDate>
<doi><json:string>10.1006/jmbi.1999.3052</json:string>
</doi>
<id>D2D35156BC771000BCDB79DD32F8B52764484B16</id>
<score>1</score>
<fulltext><json:item><extension>pdf</extension>
<original>true</original>
<mimetype>application/pdf</mimetype>
<uri>https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/fulltext.pdf</uri>
</json:item>
<json:item><extension>zip</extension>
<original>false</original>
<mimetype>application/zip</mimetype>
<uri>https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/bundle.zip</uri>
</json:item>
<istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
</titleStmt>
<publicationStmt><authority>ISTEX</authority>
<publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher>
<availability><licence><p>©1999 Academic Press</p>
</licence>
<p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p>
</availability>
<date>1999</date>
</publicationStmt>
<notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note>
<note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note>
<note>This paper is dedicated to the memory of Eladio Viñuela.</note>
<note>Edited by J. Karn</note>
<note type="content">Section title: Regular article</note>
<note type="content">Figure 1: ø29 DNA polymerase switches a DNA-paired terminus from the polymerase to the exonuclease active site without dissociating from the primer/template. (a) Analysis of dAMP incorporation onto the first complementary thymine of the template. The assay was carried out as described in Materials and Methods, using 62 pM [α-32P]dATP, 1.23 nM of non-labelled hybrid sp1/sp1c+6 and 24 nM of either mutant D12A/D66A or wild-type ø29 DNA polymerases. The mixture was incubated for ten minutes at 4 °C in the presence (lanes c; control) or absence of 70 nM of M13 ssDNA as competitor DNA. The reaction was started by adding the metal activator (c) or metal activator plus 70 nM of M13 ssDNA (rest of lanes). After incubating at 30 °C for the indicated times, samples were analysed by electrophoresis in 8 M urea-20 % polyacrylamide gels and autoradiography. (b) Exonucleolytic release of dAMP. An aliquot of each sample from the dAMP incorporation assay was analysed by thin-layer chromatography, as described in Materials and Methods. The migrating position of dAMP and the origin (DNA+non-incorporated dATP) of the chromatogram (ori) are indicated.</note>
<note type="content">Figure 2: ø29 DNA polymerase transfers the mismatched primer strand from the polymerization to the exonuclease active site without dissociating from DNA. The assay was carried out as described in Materials and Methods, using the P32-labelled hybrid molecule sp1/sp1c+18. The mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerase was incubated for ten minutes at 4 °C in the presence (c; control) or in the absence of 16 μM of non-labelled hybrid as competitor DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (lanes +). After incubation for the indicated times at 30 °C, samples were analyzed by 8 M urea-20 % polyacrylamide gel electrophoresis. The positions of the elongation products are indicated by arrows.</note>
<note type="content">Figure 3: ø29 DNA polymerase transfers the mismatched primer strand from the polymerization to the exonuclease active site without dissociating from DNA under strand displacement conditions. The assay was carried out as described in Materials and Methods, using as substrate the 5′-labelled gapped DNA hybrid molecule drawn at the top of the Figure. The mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerases and 20 μM each dATP, dTTP and dGTP, was incubated for ten minutes at 4 °C in the presence (c; control) or in the absence of 16 μM of non-labelled hybrid as competitor DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (lanes +). After incubation for the indicated times at 30 °C, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis. The positions of the elongation products are indicated by arrows.</note>
<note type="content">Figure 4: ø29 DNA polymerase transfers the 3′ terminus of the primer strand from exonuclease to polymerization active site without dissociating from DNA. The assay was carried out as described in Materials and Methods, using the hybrid sp1p/sp1c+6 as substrate. The reaction mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerases and the indicated concentration of the four dNTPs was incubated for ten minutes at 4 °C in the absence or in the presence (lane c, in this case also in the presence of 80 μM dNTPs) of 16 μM of the non-labelled hybrid as challenger DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (rest of lanes). After incubation for the indicated times at 30 °C, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis. The position of the elongation product is indicated by arrows.</note>
<note type="content">Figure 5: Schematic representation of the approach to measure the spatial coordinates (in nucleotides) of the active sites of ø29 DNA polymerase. The enzyme (grey) is represented as two structural modules, following the structural data of RB69 DNA polymerase (Wang et al., 1997): the Exo domain (left) containing the 3′-5′ exonuclease active site and the polymerization domain (right). Both exonuclease and polymerization active sites are represented as red circles. The streptavidin is represented as a red oval, while DNA is coloured blue. The portion of DNA that defines a specific channel distance (in nt) is coloured green and shown by roman letters. (a) DNA polymerase degrades the ssDNA up to a length in which streptavidin impairs the progression of exonucleolytic degradation by steric hindrance. (b) It is necessary to have a minimal distance between the polymerization active site and streptavidin to allow DNA polymerase to elongate the 3′ terminus of a primer strand. (c) DNA polymerase elongates the primer strand until it contacts the streptavidin bound to the template strand.</note>
<note type="content">Figure 6: Measurement of the distance from the 3′-5′ exonuclease active site to the surface of ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrates 5′-labelled sp1biot (ssDNA) and 5′-labelled sp1biot/sp1c+5 (dsDNA). After incubation of 12 nM of wild-type ø29 DNA polymerase for the indicated times in the absence or the presence of streptavidin, degradation of the labelled DNA was analysed by electrophoresis in 8 M urea-20 % polyacrylamide gels and autoradiography. The positions of different degradation intermediates of the DNA substrate are indicated by arrows.</note>
<note type="content">Figure 7: Measurement of the distance from the 5′-3′ polymerase active site to the surface of ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrate a collection of hybrid molecules obtained after exonucleolytic degradation of the 5′-labelled sp1biot oligo by T7 DNA polymerase, followed by hybridization to the complementary oligo sp1c+5 and subsequent incubation with streptavidin. After incubation with either wild-type or mutant D12A/D66A ø29 DNA polymerase at 4 °C for the indicated times in the absence or the presence of 80 μM dNTPs, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis and autoradiography.</note>
<note type="content">Figure 8: Measurement of the distance between the 5′-3′ polymerization active site and the entrance of the template strand in ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrate the 5′-labelled hybrid sp1/sp1c+17biot, 2 μM dNTPs and 60 nM of mutant D12A/D66A ø29 DNA polymerase. After incubation at 30 °C for the indicated times in the absence or presence of streptavidin, samples were analysed by 8 M urea-15 % polyacrylamide gel electrophoresis and autoradiography. The sizes of some elongation products are indicated.</note>
</notesStmt>
<sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
<author xml:id="author-0000"><persName><forename type="first">Miguel</forename>
<surname>de Vega</surname>
</persName>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
</author>
<author xml:id="author-0001"><persName><forename type="first">Luis</forename>
<surname>Blanco</surname>
</persName>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
</author>
<author xml:id="author-0002"><persName><forename type="first">Margarita</forename>
<surname>Salas</surname>
</persName>
<email>msalas@cbm.uam.es</email>
<note type="correspondence"><p>Corresponding author</p>
</note>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
</author>
<idno type="istex">D2D35156BC771000BCDB79DD32F8B52764484B16</idno>
<idno type="ark">ark:/67375/6H6-NC1962KS-H</idno>
<idno type="DOI">10.1006/jmbi.1999.3052</idno>
<idno type="PII">S0022-2836(99)93052-8</idno>
</analytic>
<monogr><title level="j">Journal of Molecular Biology</title>
<title level="j" type="abbrev">YJMBI</title>
<idno type="pISSN">0022-2836</idno>
<idno type="PII">S0022-2836(00)X0290-2</idno>
<imprint><publisher>ELSEVIER</publisher>
<date type="published" when="1999"></date>
<biblScope unit="volume">292</biblScope>
<biblScope unit="issue">1</biblScope>
<biblScope unit="page" from="39">39</biblScope>
<biblScope unit="page" to="51">51</biblScope>
</imprint>
</monogr>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><creation><date>1999</date>
</creation>
<langUsage><language ident="en">en</language>
</langUsage>
<abstract xml:lang="en"><p>Abstract: ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.</p>
</abstract>
<textClass><keywords scheme="keyword"><list><head>article-category</head>
<item><term>Regular article</term>
</item>
</list>
</keywords>
</textClass>
<textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head>
<item><term>ø29 DNA polymerase</term>
</item>
<item><term>primer transfer</term>
</item>
<item><term>DNA polymerase active sites</term>
</item>
</list>
</keywords>
</textClass>
<textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head>
<item><term>Pol Ik</term>
<term>Klenow fragment of DNA polymerase I from E.coli</term>
</item>
<item><term>ssDNA</term>
<term>single-stranded DNA</term>
</item>
<item><term>dsDNA double-stranded DNA</term>
</item>
<item><term>TP</term>
<term>terminal protein</term>
</item>
</list>
</keywords>
</textClass>
</profileDesc>
<revisionDesc><change when="1999-07-22">Modified</change>
<change when="1999">Published</change>
</revisionDesc>
</teiHeader>
</istex:fulltextTEI>
<json:item><extension>txt</extension>
<original>false</original>
<mimetype>text/plain</mimetype>
<uri>https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/fulltext.txt</uri>
</json:item>
</fulltext>
<metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration>
<istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity>
<istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity>
<istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity>
<istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity>
<istex:entity SYSTEM="gr5" NDATA="IMAGE" name="GR5"></istex:entity>
<istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity>
<istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity>
<istex:entity SYSTEM="gr8" NDATA="IMAGE" name="GR8"></istex:entity>
</istex:docType>
<istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid>
<aid>93052</aid>
<ce:pii>S0022-2836(99)93052-8</ce:pii>
<ce:doi>10.1006/jmbi.1999.3052</ce:doi>
<ce:copyright type="full-transfer" year="1999">Academic Press</ce:copyright>
<ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text>
</ce:doctopic>
</ce:doctopics>
</item-info>
<head><ce:article-footnote><ce:label>☆</ce:label>
<ce:note-para>This paper is dedicated to the memory of Eladio Viñuela.</ce:note-para>
</ce:article-footnote>
<ce:dochead><ce:textfn>Regular article</ce:textfn>
</ce:dochead>
<ce:title>Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup>
</ce:cross-ref>
<ce:footnote id="FN1"><ce:label>1</ce:label>
<ce:note-para><ce:bold><ce:italic>Edited by J. Karn</ce:italic>
</ce:bold>
</ce:note-para>
</ce:footnote>
</ce:title>
<ce:author-group><ce:author><ce:given-name>Miguel</ce:given-name>
<ce:surname>de Vega</ce:surname>
<ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup>
</ce:cross-ref>
</ce:author>
<ce:author><ce:given-name>Luis</ce:given-name>
<ce:surname>Blanco</ce:surname>
<ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup>
</ce:cross-ref>
</ce:author>
<ce:author><ce:given-name>Margarita</ce:given-name>
<ce:surname>Salas</ce:surname>
<ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup>
</ce:cross-ref>
<ce:cross-ref refid="COR1">*</ce:cross-ref>
<ce:e-address>msalas@cbm.uam.es</ce:e-address>
</ce:author>
<ce:affiliation id="AFF1"><ce:label>a</ce:label>
<ce:textfn>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</ce:textfn>
</ce:affiliation>
<ce:correspondence id="COR1"><ce:label>*</ce:label>
<ce:text>Corresponding author</ce:text>
</ce:correspondence>
</ce:author-group>
<ce:date-received day="26" month="5" year="1999"></ce:date-received>
<ce:date-revised day="22" month="7" year="1999"></ce:date-revised>
<ce:date-accepted day="22" month="7" year="1999"></ce:date-accepted>
<ce:abstract><ce:section-title>Abstract</ce:section-title>
<ce:abstract-sec><ce:simple-para>ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode <ce:italic>versus</ce:italic>
 the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.</ce:simple-para>
</ce:abstract-sec>
</ce:abstract>
<ce:keywords><ce:section-title>Keywords</ce:section-title>
<ce:keyword><ce:text>ø29 DNA polymerase</ce:text>
</ce:keyword>
<ce:keyword><ce:text>primer transfer</ce:text>
</ce:keyword>
<ce:keyword><ce:text>DNA polymerase active sites</ce:text>
</ce:keyword>
</ce:keywords>
<ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title>
<ce:keyword><ce:text>Pol Ik</ce:text>
<ce:keyword><ce:text>Klenow fragment of DNA polymerase I from <ce:italic>E.coli</ce:italic>
</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>ssDNA</ce:text>
<ce:keyword><ce:text>single-stranded DNA</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>dsDNA double-stranded DNA</ce:text>
</ce:keyword>
<ce:keyword><ce:text>TP</ce:text>
<ce:keyword><ce:text>terminal protein</ce:text>
</ce:keyword>
</ce:keyword>
</ce:keywords>
</head>
</converted-article>
</istex:document>
</istex:metadataXml>
<mods version="3.6"><titleInfo lang="en"><title>Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
</titleInfo>
<titleInfo type="alternative" lang="en" contentType="CDATA"><title>Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase</title>
</titleInfo>
<name type="personal"><namePart type="given">Miguel</namePart>
<namePart type="family">de Vega</namePart>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="given">Luis</namePart>
<namePart type="family">Blanco</namePart>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<name type="personal"><namePart type="given">Margarita</namePart>
<namePart type="family">Salas</namePart>
<affiliation>E-mail: msalas@cbm.uam.es</affiliation>
<affiliation>Centro de Biologı́a Molecular “Severo Ochoa” (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain</affiliation>
<description>Corresponding author</description>
<role><roleTerm type="text">author</roleTerm>
</role>
</name>
<typeOfResource>text</typeOfResource>
<genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre>
<originInfo><publisher>ELSEVIER</publisher>
<dateIssued encoding="w3cdtf">1999</dateIssued>
<dateModified encoding="w3cdtf">1999-07-22</dateModified>
<copyrightDate encoding="w3cdtf">1999</copyrightDate>
</originInfo>
<language><languageTerm type="code" authority="iso639-2b">eng</languageTerm>
<languageTerm type="code" authority="rfc3066">en</languageTerm>
</language>
<abstract lang="en">Abstract: ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.</abstract>
<note>This paper is dedicated to the memory of Eladio Viñuela.</note>
<note type="footnote">Edited by J. Karn</note>
<note type="content">Section title: Regular article</note>
<note type="content">Figure 1: ø29 DNA polymerase switches a DNA-paired terminus from the polymerase to the exonuclease active site without dissociating from the primer/template. (a) Analysis of dAMP incorporation onto the first complementary thymine of the template. The assay was carried out as described in Materials and Methods, using 62 pM [α-32P]dATP, 1.23 nM of non-labelled hybrid sp1/sp1c+6 and 24 nM of either mutant D12A/D66A or wild-type ø29 DNA polymerases. The mixture was incubated for ten minutes at 4 °C in the presence (lanes c; control) or absence of 70 nM of M13 ssDNA as competitor DNA. The reaction was started by adding the metal activator (c) or metal activator plus 70 nM of M13 ssDNA (rest of lanes). After incubating at 30 °C for the indicated times, samples were analysed by electrophoresis in 8 M urea-20 % polyacrylamide gels and autoradiography. (b) Exonucleolytic release of dAMP. An aliquot of each sample from the dAMP incorporation assay was analysed by thin-layer chromatography, as described in Materials and Methods. The migrating position of dAMP and the origin (DNA+non-incorporated dATP) of the chromatogram (ori) are indicated.</note>
<note type="content">Figure 2: ø29 DNA polymerase transfers the mismatched primer strand from the polymerization to the exonuclease active site without dissociating from DNA. The assay was carried out as described in Materials and Methods, using the P32-labelled hybrid molecule sp1/sp1c+18. The mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerase was incubated for ten minutes at 4 °C in the presence (c; control) or in the absence of 16 μM of non-labelled hybrid as competitor DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (lanes +). After incubation for the indicated times at 30 °C, samples were analyzed by 8 M urea-20 % polyacrylamide gel electrophoresis. The positions of the elongation products are indicated by arrows.</note>
<note type="content">Figure 3: ø29 DNA polymerase transfers the mismatched primer strand from the polymerization to the exonuclease active site without dissociating from DNA under strand displacement conditions. The assay was carried out as described in Materials and Methods, using as substrate the 5′-labelled gapped DNA hybrid molecule drawn at the top of the Figure. The mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerases and 20 μM each dATP, dTTP and dGTP, was incubated for ten minutes at 4 °C in the presence (c; control) or in the absence of 16 μM of non-labelled hybrid as competitor DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (lanes +). After incubation for the indicated times at 30 °C, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis. The positions of the elongation products are indicated by arrows.</note>
<note type="content">Figure 4: ø29 DNA polymerase transfers the 3′ terminus of the primer strand from exonuclease to polymerization active site without dissociating from DNA. The assay was carried out as described in Materials and Methods, using the hybrid sp1p/sp1c+6 as substrate. The reaction mixture containing 24 nM of either wild-type or mutant D12A/D66A ø29 DNA polymerases and the indicated concentration of the four dNTPs was incubated for ten minutes at 4 °C in the absence or in the presence (lane c, in this case also in the presence of 80 μM dNTPs) of 16 μM of the non-labelled hybrid as challenger DNA. The reaction was started by adding only the metal activator (lanes−and c) or metal activator plus the challenger DNA (rest of lanes). After incubation for the indicated times at 30 °C, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis. The position of the elongation product is indicated by arrows.</note>
<note type="content">Figure 5: Schematic representation of the approach to measure the spatial coordinates (in nucleotides) of the active sites of ø29 DNA polymerase. The enzyme (grey) is represented as two structural modules, following the structural data of RB69 DNA polymerase (Wang et al., 1997): the Exo domain (left) containing the 3′-5′ exonuclease active site and the polymerization domain (right). Both exonuclease and polymerization active sites are represented as red circles. The streptavidin is represented as a red oval, while DNA is coloured blue. The portion of DNA that defines a specific channel distance (in nt) is coloured green and shown by roman letters. (a) DNA polymerase degrades the ssDNA up to a length in which streptavidin impairs the progression of exonucleolytic degradation by steric hindrance. (b) It is necessary to have a minimal distance between the polymerization active site and streptavidin to allow DNA polymerase to elongate the 3′ terminus of a primer strand. (c) DNA polymerase elongates the primer strand until it contacts the streptavidin bound to the template strand.</note>
<note type="content">Figure 6: Measurement of the distance from the 3′-5′ exonuclease active site to the surface of ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrates 5′-labelled sp1biot (ssDNA) and 5′-labelled sp1biot/sp1c+5 (dsDNA). After incubation of 12 nM of wild-type ø29 DNA polymerase for the indicated times in the absence or the presence of streptavidin, degradation of the labelled DNA was analysed by electrophoresis in 8 M urea-20 % polyacrylamide gels and autoradiography. The positions of different degradation intermediates of the DNA substrate are indicated by arrows.</note>
<note type="content">Figure 7: Measurement of the distance from the 5′-3′ polymerase active site to the surface of ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrate a collection of hybrid molecules obtained after exonucleolytic degradation of the 5′-labelled sp1biot oligo by T7 DNA polymerase, followed by hybridization to the complementary oligo sp1c+5 and subsequent incubation with streptavidin. After incubation with either wild-type or mutant D12A/D66A ø29 DNA polymerase at 4 °C for the indicated times in the absence or the presence of 80 μM dNTPs, samples were analysed by 8 M urea-20 % polyacrylamide gel electrophoresis and autoradiography.</note>
<note type="content">Figure 8: Measurement of the distance between the 5′-3′ polymerization active site and the entrance of the template strand in ø29 DNA polymerase. The assay was carried out as described in Materials and Methods, using as substrate the 5′-labelled hybrid sp1/sp1c+17biot, 2 μM dNTPs and 60 nM of mutant D12A/D66A ø29 DNA polymerase. After incubation at 30 °C for the indicated times in the absence or presence of streptavidin, samples were analysed by 8 M urea-15 % polyacrylamide gel electrophoresis and autoradiography. The sizes of some elongation products are indicated.</note>
<subject><genre>article-category</genre>
<topic>Regular article</topic>
</subject>
<subject lang="en"><genre>Keywords</genre>
<topic>ø29 DNA polymerase</topic>
<topic>primer transfer</topic>
<topic>DNA polymerase active sites</topic>
</subject>
<subject lang="en"><genre>Abbreviations</genre>
<topic>Pol Ik : Klenow fragment of DNA polymerase I from E.coli</topic>
<topic>ssDNA : single-stranded DNA</topic>
<topic>dsDNA double-stranded DNA</topic>
<topic>TP : terminal protein</topic>
</subject>
<relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title>
</titleInfo>
<titleInfo type="abbreviated"><title>YJMBI</title>
</titleInfo>
<genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre>
<originInfo><publisher>ELSEVIER</publisher>
<dateIssued encoding="w3cdtf">1999</dateIssued>
</originInfo>
<identifier type="ISSN">0022-2836</identifier>
<identifier type="PII">S0022-2836(00)X0290-2</identifier>
<part><date>1999</date>
<detail type="volume"><number>292</number>
<caption>vol.</caption>
</detail>
<detail type="issue"><number>1</number>
<caption>no.</caption>
</detail>
<extent unit="issue-pages"><start>1</start>
<end>191</end>
</extent>
<extent unit="pages"><start>39</start>
<end>51</end>
</extent>
</part>
</relatedItem>
<identifier type="istex">D2D35156BC771000BCDB79DD32F8B52764484B16</identifier>
<identifier type="ark">ark:/67375/6H6-NC1962KS-H</identifier>
<identifier type="DOI">10.1006/jmbi.1999.3052</identifier>
<identifier type="PII">S0022-2836(99)93052-8</identifier>
<accessCondition type="use and reproduction" contentType="copyright">©1999 Academic Press</accessCondition>
<recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource>
<recordOrigin>Academic Press, ©1999</recordOrigin>
</recordInfo>
</mods>
<json:item><extension>json</extension>
<original>false</original>
<mimetype>application/json</mimetype>
<uri>https://api.istex.fr/ark:/67375/6H6-NC1962KS-H/record.json</uri>
</json:item>
</metadata>
<serie></serie>
</istex>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Istex/Corpus

HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002573 | SxmlIndent | more

HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 002573 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    Istex
   |étape=   Corpus
   |type=    RBID
   |clé=     ISTEX:D2D35156BC771000BCDB79DD32F8B52764484B16
   |texte=   Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase
}}

This area was generated with Dilib version V0.6.33.
Data generation: Mon Apr 20 23:26:43 2020. Site generation: Sat Mar 27 09:06:09 2021

	Serveur d'exploration MERS
	Attention, ce site est en cours de développement ! Attention, site généré par des moyens informatiques à partir de corpus bruts. Les informations ne sont donc pas validées.

Serveur d'exploration MERS

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage ø29 DNA polymerase

Source :

English descriptors

Abstract

Links to Exploration step

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri