A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction
Identifieur interne : 000E06 ( Istex/Corpus ); précédent : 000E05; suivant : 000E07A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction
Auteurs : Dominique Y. Burnouf ; Robert P. P FuchsSource :
- Mutation Research-DNA Repair [ 0921-8777 ] ; 1998.
English descriptors
- Teeft :
- Acad, Adduct, Base pairs, Biochemistry, Biol, Burnouf, Chem, Coli, Conformation, Covalent binding, Digestion, Dnasei, Dnasei cleavage, Dnasei digestion, Duplex, Escherichia, Escherichia coli, Exonuclease activity, Final concentration, Footprint, Footprint analysis, Footprinting, Fuchs, Fuchsr, Fuchsr mutation research, Guanine, Helix, Holoenzyme, Hypersensitive, Hypersensitive sites, Kinetic experiments, Klenow fragment, Lesion, Model junction, Molecular dynamics, Monomodified, Monomodified duplex, Mutation, Natl, Nucleotide, Oligonucleotide, Phosphodiester, Phosphodiester bonds, Phosphodiester bounds, Polymerase, Polymerase binding, Polymerase cleft, Polymerase holoenzyme, Primer, Proc, Replication, Same position, Template strand, Unmodified, Unmodified duplex, Unmodified duplexes.
Abstract
Abstract: Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.
Url:
DOI: 10.1016/S0921-8777(97)00058-X
Links to Exploration step
ISTEX:41F5B8C605DEE736A1A79206463C18CF321E641FLe document en format XML
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P Fuchs</name><affiliation><mods:affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:41F5B8C605DEE736A1A79206463C18CF321E641F</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1016/S0921-8777(97)00058-X</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-FMNSS9ST-4/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000E06</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000E06</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</title><author><name sortKey="Burnouf, Dominique Y" sort="Burnouf, Dominique Y" uniqKey="Burnouf D" first="Dominique Y" last="Burnouf">Dominique Y. Burnouf</name><affiliation><mods:affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</mods:affiliation></affiliation></author><author><name sortKey="Fuchs, Robert P P" sort="Fuchs, Robert P P" uniqKey="Fuchs R" first="Robert P. P" last="Fuchs">Robert P. P Fuchs</name><affiliation><mods:affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Mutation Research-DNA Repair</title><title level="j" type="abbrev">MUTDNA</title><idno type="ISSN">0921-8777</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">407</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="35">35</biblScope><biblScope unit="page" to="45">45</biblScope></imprint><idno type="ISSN">0921-8777</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0921-8777</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acad</term><term>Adduct</term><term>Base pairs</term><term>Biochemistry</term><term>Biol</term><term>Burnouf</term><term>Chem</term><term>Coli</term><term>Conformation</term><term>Covalent binding</term><term>Digestion</term><term>Dnasei</term><term>Dnasei cleavage</term><term>Dnasei digestion</term><term>Duplex</term><term>Escherichia</term><term>Escherichia coli</term><term>Exonuclease activity</term><term>Final concentration</term><term>Footprint</term><term>Footprint analysis</term><term>Footprinting</term><term>Fuchs</term><term>Fuchsr</term><term>Fuchsr mutation research</term><term>Guanine</term><term>Helix</term><term>Holoenzyme</term><term>Hypersensitive</term><term>Hypersensitive sites</term><term>Kinetic experiments</term><term>Klenow fragment</term><term>Lesion</term><term>Model junction</term><term>Molecular dynamics</term><term>Monomodified</term><term>Monomodified duplex</term><term>Mutation</term><term>Natl</term><term>Nucleotide</term><term>Oligonucleotide</term><term>Phosphodiester</term><term>Phosphodiester bonds</term><term>Phosphodiester bounds</term><term>Polymerase</term><term>Polymerase binding</term><term>Polymerase cleft</term><term>Polymerase holoenzyme</term><term>Primer</term><term>Proc</term><term>Replication</term><term>Same position</term><term>Template strand</term><term>Unmodified</term><term>Unmodified duplex</term><term>Unmodified duplexes</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>polymerase</json:string><json:string>dnasei</json:string><json:string>adduct</json:string><json:string>mutation</json:string><json:string>duplex</json:string><json:string>footprint</json:string><json:string>digestion</json:string><json:string>primer</json:string><json:string>biol</json:string><json:string>burnouf</json:string><json:string>natl</json:string><json:string>proc</json:string><json:string>acad</json:string><json:string>helix</json:string><json:string>hypersensitive</json:string><json:string>coli</json:string><json:string>footprinting</json:string><json:string>fuchsr</json:string><json:string>fuchsr mutation research</json:string><json:string>dnasei digestion</json:string><json:string>hypersensitive sites</json:string><json:string>unmodified</json:string><json:string>holoenzyme</json:string><json:string>phosphodiester</json:string><json:string>lesion</json:string><json:string>biochemistry</json:string><json:string>monomodified</json:string><json:string>guanine</json:string><json:string>oligonucleotide</json:string><json:string>chem</json:string><json:string>escherichia coli</json:string><json:string>escherichia</json:string><json:string>unmodified duplex</json:string><json:string>replication</json:string><json:string>polymerase binding</json:string><json:string>nucleotide</json:string><json:string>same position</json:string><json:string>monomodified duplex</json:string><json:string>phosphodiester bonds</json:string><json:string>base pairs</json:string><json:string>unmodified duplexes</json:string><json:string>template strand</json:string><json:string>polymerase cleft</json:string><json:string>molecular dynamics</json:string><json:string>dnasei cleavage</json:string><json:string>phosphodiester bounds</json:string><json:string>model junction</json:string><json:string>klenow fragment</json:string><json:string>footprint analysis</json:string><json:string>polymerase holoenzyme</json:string><json:string>final concentration</json:string><json:string>covalent binding</json:string><json:string>exonuclease activity</json:string><json:string>kinetic experiments</json:string><json:string>conformation</json:string><json:string>fuchs</json:string></teeft></keywords><author><json:item><name>Dominique Y Burnouf</name><affiliations><json:string>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</json:string></affiliations></json:item><json:item><name>Robert P.P Fuchs</name><affiliations><json:string>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>T7 DNA polymerase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AAF</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Single DNA adduct</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNAseI footprint</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA polymerase blocking lesion</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AAF, N-2-acetylaminofluorene</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AF, N-2-aminofluorene</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNaseI, bovine pancreatic deoxyribonuclease I</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HIV-RT, Human immunodeficiency virus reverse transcriptase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Tris, tris(hydroxymethyl)aminomethane</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>dNTP, deoxynucleoside 5′-triphosphate</value></json:item></subject><arkIstex>ark:/67375/6H6-FMNSS9ST-4</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.</abstract><qualityIndicators><score>8.884</score><pdfWordCount>5354</pdfWordCount><pdfCharCount>32822</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>540 x 712 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>157</abstractWordCount><abstractCharCount>1096</abstractCharCount><keywordCount>11</keywordCount></qualityIndicators><title>A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</title><pmid><json:string>9539979</json:string></pmid><pii><json:string>S0921-8777(97)00058-X</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Mutation Research-DNA Repair</title><language><json:string>unknown</json:string></language><publicationDate>1998</publicationDate><issn><json:string>0921-8777</json:string></issn><pii><json:string>S0921-8777(00)X0024-9</json:string></pii><volume>407</volume><issue>1</issue><pages><first>35</first><last>45</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1998</json:string></date><geogName></geogName><orgName><json:string>Biochem</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>M. Bichara</json:string><json:string>A.M. Freund</json:string><json:string>B.M. Alberts</json:string><json:string>G. Kamer</json:string><json:string>D. Goodsell</json:string><json:string>Janet Lindsley</json:string><json:string>P. Argos</json:string><json:string>K.A. Johnson</json:string><json:string>G. Villani</json:string><json:string>G.P. Lomonossoff</json:string><json:string>S. Broyde</json:string><json:string>L.A. Blanco</json:string><json:string>R. Xu</json:string><json:string>J. Biol</json:string><json:string>J.S. Hoffmann</json:string><json:string>M. O’Donnell</json:string><json:string>N. Koffel-Schwartz</json:string><json:string>J.A. Reems</json:string><json:string>Y. Burnouf</json:string><json:string>O. Poch</json:string><json:string>M. Widdifield</json:string><json:string>K.B. Lipkowitz</json:string><json:string>G.S. Le</json:string><json:string>T.A. Steitz</json:string><json:string>R.P. Fuchs</json:string><json:string>E. Seeberg</json:string><json:string>S.F. O’Handley</json:string><json:string>E. Delagoutte</json:string><json:string>J.E. Leclerc</json:string><json:string>J. Huan</json:string><json:string>J.E. LeClerc</json:string><json:string>D. Burnouf</json:string><json:string>L.S. Beese</json:string><json:string>P. Clark</json:string><json:string>P. Valladier-Belguise</json:string><json:string>B.V. Keywords</json:string><json:string>E. Arnold</json:string><json:string>R.L. Williams</json:string><json:string>C. Lesca</json:string><json:string>J. Wang</json:string><json:string>B.M. Wohrl</json:string><json:string>M. Leng</json:string><json:string>M.M. Georgiadis</json:string><json:string>T.A. Kunkel</json:string><json:string>R.P.P. Fuchs</json:string><json:string>X. Lu</json:string><json:string>M.P. Daune</json:string><json:string>D. Moras</json:string><json:string>M.J. Donlin</json:string><json:string>T.R. Krugh</json:string><json:string>Kenneth Johnson</json:string><json:string>R.L. Napolitano</json:string><json:string>H.R. Drew</json:string><json:string>B.E. Hingerty</json:string><json:string>P.J. Butler</json:string><json:string>P. Koehl</json:string><json:string>A.J. Clark</json:string><json:string>A.L. Ferris</json:string><json:string>E.C. Miller</json:string><json:string>M. Delarue</json:string><json:string>H. Maki</json:string><json:string>C.S. McHenry</json:string><json:string>J. Turner</json:string><json:string>X. Veaute</json:string><json:string>P.P. Fuchs</json:string><json:string>H. Heumann</json:string><json:string>J. Pouyet</json:string><json:string>I. Wong</json:string><json:string>D. Suck</json:string><json:string>Sydney Edwards</json:string><json:string>Alan R. Liss</json:string><json:string>J. Mol</json:string><json:string>M.L. Kopka</json:string><json:string>R.G. Nanni</json:string><json:string>J.A. Miller</json:string><json:string>M.M. Munn</json:string><json:string>M. Defais</json:string><json:string>N. Tordo</json:string><json:string>A. Hizi</json:string><json:string>C. Oefner</json:string><json:string>R.E. Dickerson</json:string><json:string>N.D.F. Grindley</json:string><json:string>J.E. Lindsley</json:string><json:string>S.K. Banerjee</json:string><json:string>T. Hermann</json:string><json:string>G. Dirheimer</json:string><json:string>S.H. Hughes</json:string><json:string>J.M. Friedman</json:string><json:string>D.L. Ollis</json:string><json:string>G.C. Walker</json:string><json:string>E. Bertrant-Burgraff</json:string><json:string>A. Klug</json:string><json:string>I.B. Lambert</json:string><json:string>J.M. Verdier</json:string><json:string>D.Y. Burnouf</json:string><json:string>J. Gangloff</json:string><json:string>O. Schatz</json:string><json:string>J. Natl</json:string><json:string>C.M. Joyce</json:string><json:string>A. Telesnitsky</json:string><json:string>M.A. Blasco</json:string><json:string>D.C. Thomas</json:string><json:string>N. Schwartz</json:string><json:string>M. Daune</json:string><json:string>J. Pillaire</json:string><json:string>W. Metzger</json:string><json:string>K.J. Hacker</json:string><json:string>W.A. Hendrickson</json:string><json:string>S. Murli</json:string><json:string>P. Rio</json:string><json:string>M. Salas</json:string><json:string>M. Sekiguchi</json:string><json:string>J. Dunan</json:string><json:string>A.A. Travers</json:string><json:string>C. Tantillo</json:string><json:string>P.T. Stukenberg</json:string><json:string>A. Borden</json:string><json:string>J. Ding</json:string><json:string>F. Coin</json:string><json:string>F.A. Beland</json:string><json:string>L.A. Kohlstaedt</json:string><json:string>M. Ptak</json:string><json:string>D. Kern</json:string><json:string>P. Pjura</json:string><json:string>S.S. Patel</json:string><json:string>R.B. Christensen</json:string><json:string>C.C. Lester</json:string><json:string>M.A. Jacobo-Mollina</json:string><json:string>P.A. Rice</json:string><json:string>W.H. Konigsberg</json:string><json:string>P. Belguise-Valladier</json:string><json:string>C.W. Lawrence</json:string><json:string>D.G. Sanford</json:string></persName><placeName><json:string>York</json:string><json:string>Derbyshire</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Gangloff et al.</json:string><json:string>Gene 1991</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-FMNSS9ST-4</json:string></ark><categories><wos></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - developmental 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 - Genetics</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Toxicology</json:string><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></inist></categories><publicationDate>1998</publicationDate><copyrightDate>1998</copyrightDate><doi><json:string>10.1016/S0921-8777(97)00058-X</json:string></doi><id>41F5B8C605DEE736A1A79206463C18CF321E641F</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-FMNSS9ST-4/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-FMNSS9ST-4/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-FMNSS9ST-4/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1998 Elsevier Science B.V.</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1998</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 type="content">Fig. 1: Fifteen percent PAGE analysis of the DNaseI digestion pattern of 5′P32 labelled 30/44 duplex bound to T7 (exo−) DNA polymerase. Lane 1 and 2: unmodified duplex, lane 3 and 4: AAF monomodified duplex. For easier comparison between the bands, polymerase was added in reactions shown in lane 2 and 3. The digestion profiles of naked unmodified and AAF modified duplexes are displayed in lane 1 and 4, respectively. Numberings are indicated to the right. Boxes on each side of the gel indicate the position of protected regions (light striped boxes P1 and P2) and hypersensitive sites (dark bars) on unmodified (left) and AAF modified (right) duplexes.</note><note type="content">Fig. 2: Fifteen percent PAGE analysis of the DNaseI digestion pattern of 5′P32 labelled 44/30 duplex bound to T7 (exo−) DNA polymerase. Lane 1 and 2: unmodified duplex, lane 3 and 4: AAF monomodified duplex at position G14. Lane 2 and 3: footprints experiments were done in presence of T7 (exo−) DNA polymerase. Lane 1 and 4: no polymerase added. Numberings are indicated to the right. Boxes on each side of the gel indicate the position of protected regions (light striped boxes P1 and P2) and hypersensitive sites (dark bars) on unmodified (left) and AAF modified (right) duplexes. The grey box visualizes the AAF protection toward DNaseI digestion. Arrows indicate the extend of double strand and single strand regions of the duplexes.</note><note type="content">Fig. 3: Schematic representation of the footprinting data. (A) T7 (exo−) DNA polymerase bound to unmodified primer–template duplex. (B) T7 (exo−) DNA polymerase bound to AAF-modified primer–template duplex. Open boxes represent the P1 and P2 protected regions. Black bars indicate the hypersensitive sites. The hatched box visualizes the AAF protection towards DNAseI digestion. The AAF moiety is represented in grey, stacked on the adjacent base pair, while the modified guanine is displaced outside the helix. Grey lines represent phosphodiester bounds displaying an identical sensitivity toward DNaseI digestion in absence or presence of the polymerase. Black triangles represent sites unsensitive to DNase I digestion. In (A), the heights of the black boxes correlates with the intensity of DNaseI cutting (see Figs. 1 and 2).</note><note type="content">Fig. 4: PAGE analysis of elongation products of 5′P32 labelled 25 mer primer/44 mer template (100 nM), by T7 (exo−) DNA polymerase (300 nM). Only dCTP, dGTP and dTTP were added in the reaction mix (200 mM each). Reactions were stopped at indicated time by addition of EDTA (0.3 M final concentration). The scheme above the picture describes the elongation experiment. Positions of primer and elongated products are indicated to the right. Radioactive band at time 0 represents the 25 mer primer. Note that most of it (about 90%) is elongated in the initial step of the experiment, and that the remaining material (about 10%) is constant during the time course, indicating that the elongation is processive.</note><note type="content">Fig. 5: Schematic 2D representation of the footprinting data on a model B-DNA conformation. (A) T7 (exo−) DNA polymerase bound to unmodified primer–template duplex. (B) T7 (exo−) DNA polymerase bound to AAF-modified primer–template duplex. Dots represent the nucleotides and lines shown at selected positions represent the phosphodiester bounds. Arrows indicate the 5′–3′ polarity of each strand. Grey dots visualize an equal sensitivity of the phosphodiester bounds to DNaseI in absence or presence of the polymerase; black dots represent insensitive sites and open dots indicate positions that have not been analyzed. Black or grey squares indicate the hypersensitive positions and open squares, the P1 and P2 protected area; open triangles represent the AAF induced protection and the black triangle indicates the site of modification.</note><note type="content">Table 1: Synthetic duplexes used in the primer elongation (25/44 mer) and footprinting experiments (30/44 mer)</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</title><author xml:id="author-0000"><persName><forename type="first">Dominique Y</forename><surname>Burnouf</surname></persName><affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Robert P.P</forename><surname>Fuchs</surname></persName><affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</affiliation></author><idno type="istex">41F5B8C605DEE736A1A79206463C18CF321E641F</idno><idno type="ark">ark:/67375/6H6-FMNSS9ST-4</idno><idno type="DOI">10.1016/S0921-8777(97)00058-X</idno><idno type="PII">S0921-8777(97)00058-X</idno></analytic><monogr><title level="j">Mutation Research-DNA Repair</title><title level="j" type="abbrev">MUTDNA</title><idno type="pISSN">0921-8777</idno><idno type="PII">S0921-8777(00)X0024-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">407</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="35">35</biblScope><biblScope unit="page" to="45">45</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1998</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>T7 DNA polymerase</term></item><item><term>AAF</term></item><item><term>Single DNA adduct</term></item><item><term>DNAseI footprint</term></item><item><term>DNA polymerase blocking lesion</term></item></list></keywords></textClass><textClass><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>AAF, N-2-acetylaminofluorene</term></item><item><term>AF, N-2-aminofluorene</term></item><item><term>DNaseI, bovine pancreatic deoxyribonuclease I</term></item><item><term>HIV-RT, Human immunodeficiency virus reverse transcriptase</term></item><item><term>Tris, tris(hydroxymethyl)aminomethane</term></item><item><term>dNTP, deoxynucleoside 5′-triphosphate</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1997-10-28">Modified</change><change when="1998">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-FMNSS9ST-4/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:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>MUTDNA</jid><aid>6819</aid><ce:pii>S0921-8777(97)00058-X</ce:pii><ce:doi>10.1016/S0921-8777(97)00058-X</ce:doi><ce:copyright year="1998" type="full-transfer">Elsevier Science B.V.</ce:copyright></item-info><head><ce:title>A single <ce:italic>N</ce:italic>-2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</ce:title><ce:author-group><ce:author><ce:given-name>Dominique Y</ce:given-name><ce:surname>Burnouf</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>Robert P.P</ce:given-name><ce:surname>Fuchs</ce:surname></ce:author><ce:affiliation><ce:textfn>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +33-03-88-11-90-31; fax: +33-03-88-11-90-98; E-mail: dominique.burnouf@ircad.u-strabg.fr.</ce:text></ce:correspondence></ce:author-group><ce:date-received day="21" month="8" year="1997"></ce:date-received><ce:date-revised day="28" month="10" year="1997"></ce:date-revised><ce:date-accepted day="31" month="10" year="1997"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a <ce:italic>N</ce:italic>-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>T7 DNA polymerase</ce:text></ce:keyword><ce:keyword><ce:text>AAF</ce:text></ce:keyword><ce:keyword><ce:text>Single DNA adduct</ce:text></ce:keyword><ce:keyword><ce:text>DNAseI footprint</ce:text></ce:keyword><ce:keyword><ce:text>DNA polymerase blocking lesion</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>AAF, <ce:italic>N</ce:italic>-2-acetylaminofluorene</ce:text></ce:keyword><ce:keyword><ce:text>AF, <ce:italic>N</ce:italic>-2-aminofluorene</ce:text></ce:keyword><ce:keyword><ce:text>DNaseI, bovine pancreatic deoxyribonuclease I</ce:text></ce:keyword><ce:keyword><ce:text>HIV-RT, Human immunodeficiency virus reverse transcriptase</ce:text></ce:keyword><ce:keyword><ce:text>Tris, tris(hydroxymethyl)aminomethane</ce:text></ce:keyword><ce:keyword><ce:text>dNTP, deoxynucleoside 5′-triphosphate</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>A single N -2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>A single</title></titleInfo><name type="personal"><namePart type="given">Dominique Y</namePart><namePart type="family">Burnouf</namePart><affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</affiliation><description>Corresponding author. Tel.: +33-03-88-11-90-31; fax: +33-03-88-11-90-98; E-mail: dominique.burnouf@ircad.u-strabg.fr.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Robert P.P</namePart><namePart type="family">Fuchs</namePart><affiliation>UPR 9003, Cancérogénèse et Mutagénèse Moléculaire et Structurale, CNRS, Laboratoire d'Epidémiologie Moléculaire du Cancer, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67091, Strasbourg, France</affiliation><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">1998</dateIssued><dateModified encoding="w3cdtf">1997-10-28</dateModified><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo−) DNA polymerase bound to a model primer–template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer–template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.</abstract><note type="content">Fig. 1: Fifteen percent PAGE analysis of the DNaseI digestion pattern of 5′P32 labelled 30/44 duplex bound to T7 (exo−) DNA polymerase. Lane 1 and 2: unmodified duplex, lane 3 and 4: AAF monomodified duplex. For easier comparison between the bands, polymerase was added in reactions shown in lane 2 and 3. The digestion profiles of naked unmodified and AAF modified duplexes are displayed in lane 1 and 4, respectively. Numberings are indicated to the right. Boxes on each side of the gel indicate the position of protected regions (light striped boxes P1 and P2) and hypersensitive sites (dark bars) on unmodified (left) and AAF modified (right) duplexes.</note><note type="content">Fig. 2: Fifteen percent PAGE analysis of the DNaseI digestion pattern of 5′P32 labelled 44/30 duplex bound to T7 (exo−) DNA polymerase. Lane 1 and 2: unmodified duplex, lane 3 and 4: AAF monomodified duplex at position G14. Lane 2 and 3: footprints experiments were done in presence of T7 (exo−) DNA polymerase. Lane 1 and 4: no polymerase added. Numberings are indicated to the right. Boxes on each side of the gel indicate the position of protected regions (light striped boxes P1 and P2) and hypersensitive sites (dark bars) on unmodified (left) and AAF modified (right) duplexes. The grey box visualizes the AAF protection toward DNaseI digestion. Arrows indicate the extend of double strand and single strand regions of the duplexes.</note><note type="content">Fig. 3: Schematic representation of the footprinting data. (A) T7 (exo−) DNA polymerase bound to unmodified primer–template duplex. (B) T7 (exo−) DNA polymerase bound to AAF-modified primer–template duplex. Open boxes represent the P1 and P2 protected regions. Black bars indicate the hypersensitive sites. The hatched box visualizes the AAF protection towards DNAseI digestion. The AAF moiety is represented in grey, stacked on the adjacent base pair, while the modified guanine is displaced outside the helix. Grey lines represent phosphodiester bounds displaying an identical sensitivity toward DNaseI digestion in absence or presence of the polymerase. Black triangles represent sites unsensitive to DNase I digestion. In (A), the heights of the black boxes correlates with the intensity of DNaseI cutting (see Figs. 1 and 2).</note><note type="content">Fig. 4: PAGE analysis of elongation products of 5′P32 labelled 25 mer primer/44 mer template (100 nM), by T7 (exo−) DNA polymerase (300 nM). Only dCTP, dGTP and dTTP were added in the reaction mix (200 mM each). Reactions were stopped at indicated time by addition of EDTA (0.3 M final concentration). The scheme above the picture describes the elongation experiment. Positions of primer and elongated products are indicated to the right. Radioactive band at time 0 represents the 25 mer primer. Note that most of it (about 90%) is elongated in the initial step of the experiment, and that the remaining material (about 10%) is constant during the time course, indicating that the elongation is processive.</note><note type="content">Fig. 5: Schematic 2D representation of the footprinting data on a model B-DNA conformation. (A) T7 (exo−) DNA polymerase bound to unmodified primer–template duplex. (B) T7 (exo−) DNA polymerase bound to AAF-modified primer–template duplex. Dots represent the nucleotides and lines shown at selected positions represent the phosphodiester bounds. Arrows indicate the 5′–3′ polarity of each strand. Grey dots visualize an equal sensitivity of the phosphodiester bounds to DNaseI in absence or presence of the polymerase; black dots represent insensitive sites and open dots indicate positions that have not been analyzed. Black or grey squares indicate the hypersensitive positions and open squares, the P1 and P2 protected area; open triangles represent the AAF induced protection and the black triangle indicates the site of modification.</note><note type="content">Table 1: Synthetic duplexes used in the primer elongation (25/44 mer) and footprinting experiments (30/44 mer)</note><subject><genre>Keywords</genre><topic>T7 DNA polymerase</topic><topic>AAF</topic><topic>Single DNA adduct</topic><topic>DNAseI footprint</topic><topic>DNA polymerase blocking lesion</topic></subject><subject><genre>Abbreviations</genre><topic>AAF, N-2-acetylaminofluorene</topic><topic>AF, N-2-aminofluorene</topic><topic>DNaseI, bovine pancreatic deoxyribonuclease I</topic><topic>HIV-RT, Human immunodeficiency virus reverse transcriptase</topic><topic>Tris, tris(hydroxymethyl)aminomethane</topic><topic>dNTP, deoxynucleoside 5′-triphosphate</topic></subject><relatedItem type="host"><titleInfo><title>Mutation Research-DNA Repair</title></titleInfo><titleInfo type="abbreviated"><title>MUTDNA</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">1998</dateIssued></originInfo><identifier type="ISSN">0921-8777</identifier><identifier type="PII">S0921-8777(00)X0024-9</identifier><part><date>1998</date><detail type="volume"><number>407</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>86</end></extent><extent unit="pages"><start>35</start><end>45</end></extent></part></relatedItem><identifier type="istex">41F5B8C605DEE736A1A79206463C18CF321E641F</identifier><identifier type="ark">ark:/67375/6H6-FMNSS9ST-4</identifier><identifier type="DOI">10.1016/S0921-8777(97)00058-X</identifier><identifier type="PII">S0921-8777(97)00058-X</identifier><accessCondition type="use and reproduction" contentType="copyright">©1998 Elsevier Science B.V.</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>Elsevier Science B.V., ©1998</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-FMNSS9ST-4/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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