Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†
Identifieur interne : 000D73 ( Istex/Corpus ); précédent : 000D72; suivant : 000D74Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†
Auteurs : Mi-Sook Kim ; F. Peter GuengerichSource :
- Chemical Research in Toxicology [ 0893-228x ] ; 1998.
Abstract
The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
Url:
DOI: 10.1021/tx970206m
Links to Exploration step
ISTEX:B587D6D00D21F938208F695EFBADD5C3568FA782Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†</title><author><name sortKey="Kim, Mi Sook" sort="Kim, Mi Sook" uniqKey="Kim M" first="Mi-Sook" last="Kim">Mi-Sook Kim</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Merck & Company, P.O. Box 2000,126 EastLincoln Ave., Rahway, NJ 07065-0900.</mods:affiliation></affiliation></author><author><name sortKey="Guengerich, F Peter" sort="Guengerich, F Peter" uniqKey="Guengerich F" first="F. Peter" last="Guengerich">F. Peter Guengerich</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Address correspondence to this author. Tel: (615) 322-2261.Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:B587D6D00D21F938208F695EFBADD5C3568FA782</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1021/tx970206m</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000D73</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000D73</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Polymerase Blockage and Misincorporation of dNTPs
Opposite the Ethylene Dibromide-Derived DNA Adducts
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-Guanyl)ethyl]glutathione,
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Guanyl)ethyl]glutathione, and
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-Guanyl)ethyl]glutathione<ref type="bib" target="#tx970206mAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Kim, Mi Sook" sort="Kim, Mi Sook" uniqKey="Kim M" first="Mi-Sook" last="Kim">Mi-Sook Kim</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Merck & Company, P.O. Box 2000,126 EastLincoln Ave., Rahway, NJ 07065-0900.</mods:affiliation></affiliation></author><author><name sortKey="Guengerich, F Peter" sort="Guengerich, F Peter" uniqKey="Guengerich F" first="F. Peter" last="Guengerich">F. Peter Guengerich</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Address correspondence to this author. Tel: (615) 322-2261.Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Chemical Research in Toxicology</title><title level="j" type="abbrev">Chem. Res. Toxicol.</title><idno type="ISSN">0893-228x</idno><idno type="eISSN">1520-5010</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1998</date><date type="published">1998</date><biblScope unit="vol">11</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="311">311</biblScope><biblScope unit="page" to="316">316</biblScope></imprint><idno type="ISSN">0893-228x</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0893-228x</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>polymerase</json:string><json:string>adduct</json:string><json:string>dctp</json:string><json:string>datp</json:string><json:string>dttp</json:string><json:string>primer</json:string><json:string>dgtp</json:string><json:string>guengerich</json:string><json:string>chem</json:string><json:string>mutation</json:string><json:string>coli</json:string><json:string>toxicol</json:string><json:string>biochemistry</json:string><json:string>ethylene</json:string><json:string>misinsertion</json:string><json:string>dntps</json:string><json:string>misincorporation</json:string><json:string>escherichia</json:string><json:string>dntp</json:string><json:string>kcat</json:string><json:string>misinsertion ratio</json:string><json:string>pairing</json:string><json:string>dttp incorporation</json:string><json:string>dibromide</json:string><json:string>miscoding</json:string><json:string>oligonucleotides</json:string><json:string>replication</json:string><json:string>ethylene dibromide</json:string><json:string>incorporation</json:string><json:string>sequence analysis</json:string><json:string>dctp incorporation</json:string><json:string>human immunodeficiency</json:string><json:string>derivative</json:string><json:string>insertion</json:string><json:string>escherichia coli polymerase</json:string><json:string>kinetic analysis</json:string><json:string>adducted template</json:string><json:string>klenow fragment</json:string><json:string>nucleotide sequence analysis</json:string><json:string>misincorporation frequency</json:string><json:string>datp opposite</json:string><json:string>dttp opposite</json:string><json:string>primer extension reaction</json:string><json:string>ethylating agent</json:string><json:string>nucleotide</json:string></teeft></keywords><author><json:item><name>KIM Mi-Sook</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</json:string><json:string>Current address: Merck & Company, P.O. Box 2000,126 EastLincoln Ave., Rahway, NJ 07065-0900.</json:string></affiliations></json:item><json:item><name>GUENGERICH F. Peter</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</json:string><json:string>Address correspondence to this author. Tel: (615) 322-2261.Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-QTD464B6-M</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.</abstract><qualityIndicators><score>9.094</score><pdfWordCount>4742</pdfWordCount><pdfCharCount>31345</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>6</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>790</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>196</abstractWordCount><abstractCharCount>1568</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†</title><genre><json:string>research-article</json:string></genre><host><title>Chemical Research in Toxicology</title><language><json:string>unknown</json:string></language><issn><json:string>0893-228x</json:string></issn><eissn><json:string>1520-5010</json:string></eissn><volume>11</volume><issue>4</issue><pages><first>311</first><last>316</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-QTD464B6-M</json:string></ark><categories><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>10.1021/tx970206m</json:string></doi><id>B587D6D00D21F938208F695EFBADD5C3568FA782</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Polymerase Blockage and Misincorporation of dNTPs
Opposite the Ethylene Dibromide-Derived DNA Adducts
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-Guanyl)ethyl]glutathione,
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Guanyl)ethyl]glutathione, and
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-Guanyl)ethyl]glutathione<ref type="bib" target="#tx970206mAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 1998 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">1998</date><date type="Copyright" when="1998">1998</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Polymerase Blockage and Misincorporation of dNTPs
Opposite the Ethylene Dibromide-Derived DNA Adducts
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-Guanyl)ethyl]glutathione,
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Guanyl)ethyl]glutathione, and
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-Guanyl)ethyl]glutathione<ref type="bib" target="#tx970206mAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Kim</surname><forename type="first">Mi-Sook</forename></persName><affiliation><orgName type="institution">Department of Biochemistry and Center in Molecular Toxicology</orgName><orgName type="institution">Vanderbilt University School of Medicine</orgName><address><addrLine>Nashville</addrLine><addrLine>Tennessee 37232-0146</addrLine></address></affiliation><note place="foot" n="tx970206mAF4"><ref>§</ref><p>
Current address: Merck & Company, P.O. Box 2000,
126 East
Lincoln Ave., Rahway, NJ 07065-0900.</p></note></author><author xml:id="author-0001" role="corresp"><persName><surname>Guengerich</surname><forename type="first">F. Peter</forename></persName><affiliation><orgName type="institution">Department of Biochemistry and Center in Molecular Toxicology</orgName><orgName type="institution">Vanderbilt University School of Medicine</orgName><address><addrLine>Nashville</addrLine><addrLine>Tennessee 37232-0146</addrLine></address></affiliation><affiliation role="corresp"> Address correspondence to this author. Tel: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</affiliation></author><idno type="istex">B587D6D00D21F938208F695EFBADD5C3568FA782</idno><idno type="ark">ark:/67375/TPS-QTD464B6-M</idno><idno type="DOI">10.1021/tx970206m</idno></analytic><monogr><title level="j" type="main">Chemical Research in Toxicology</title><title level="j" type="abbrev">Chem. Res. Toxicol.</title><idno type="acspubs">tx</idno><idno type="coden">crtoec</idno><idno type="pISSN">0893-228x</idno><idno type="eISSN">1520-5010</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1998</date><date type="published">1998</date><biblScope unit="vol">11</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="311">311</biblScope><biblScope unit="page" to="316">316</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>The carcinogen ethylene dibromide (EDB) has been shown to cause
glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions,
in a variety of bacterial
and eukaryotic systems. The known DNA adducts
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-guanyl)ethyl]GSH,
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-guanyl)ethyl]GSH, and
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-guanyl)ethyl]GSH
were individually placed at a site in a single
oligonucleotide. Polymerase extension studies were carried out
using <hi rend="italic">Escherichia coli</hi>
polymerase I exo<hi rend="superscript">-</hi> (Klenow fragment, Kf<hi rend="superscript">-</hi>)
and polymerase II exo<hi rend="superscript">-</hi> (pol II<hi rend="superscript">-</hi>),
bacteriophage T7
polymerase exo<hi rend="superscript">-</hi>, and human immunodeficiency virus-1
reverse transcriptase in order to
characterize misincorporation events. Even though extension was
not as efficient as with the
nonadducted template, some fully extended primers were observed with
the template containing
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-guanyl)ethyl]GSH
using all of these polymerases. dCTP was the most
preferred
nucleotide incorporated opposite
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-guanyl)ethyl]GSH
by most of polymerases examined;
however, dTTP incorporation was observed opposite
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">7</hi>-guanyl)ethyl]GSH
with pol II<hi rend="superscript">-</hi>.
Both
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-guanyl)ethyl]GSH
and
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-guanyl)ethyl]GSH
strongly blocked replication
by all polymerases. Only dATP and dGTP were incorporated opposite
<hi rend="italic">S</hi>-[2-(<hi rend="italic">N</hi><hi rend="superscript">2</hi>-guanyl)ethyl]GSH by both Kf<hi rend="superscript">-</hi> and pol II<hi rend="superscript">-</hi>.
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-Guanyl)ethyl]GSH
was shown to strongly code for dATP
incorporation by Kf<hi rend="superscript">-</hi>. With pol II<hi rend="superscript">-</hi>,
dTTP was incorporated opposite
<hi rend="italic">S</hi>-[2-(<hi rend="italic">O</hi><hi rend="superscript">6</hi>-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the
carcinogen EDB blocked
the polymerases and were capable of miscoding.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">tx</journal-id><journal-id journal-id-type="coden">crtoec</journal-id><journal-title-group><journal-title>Chemical Research in Toxicology</journal-title><abbrev-journal-title>Chem. Res. Toxicol.</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0893-228x</issn><issn pub-type="epub">1520-5010</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/crt</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/tx970206m</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Polymerase Blockage and Misincorporation of dNTPs
Opposite the Ethylene Dibromide-Derived DNA Adducts
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-Guanyl)ethyl]glutathione,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-Guanyl)ethyl]glutathione, and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-Guanyl)ethyl]glutathione<xref rid="tx970206mAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Kim</surname><given-names>Mi-Sook</given-names></name><xref rid="tx970206mAF4"><sup>§</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Guengerich</surname><given-names>F. Peter</given-names></name><xref rid="tx970206mAF1">*</xref></contrib><aff>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232-0146
</aff></contrib-group><author-notes><fn id="tx970206mAF4"><label>§</label><p>
Current address: Merck & Company, P.O. Box 2000,
126 East
Lincoln Ave., Rahway, NJ 07065-0900.</p></fn><corresp id="tx970206mAF1">
Address correspondence to this author. Tel: (615) 322-2261.
Fax:
(615) 322-3141. E-mail:
guengerich@toxicology.mc.vanderbilt.edu.</corresp></author-notes><pub-date pub-type="epub"><day>26</day><month>02</month><year>1998</year></pub-date><pub-date pub-type="ppub"><day>20</day><month>04</month><year>1998</year></pub-date><volume>11</volume><issue>4</issue><fpage>311</fpage><lpage>316</lpage><supplementary-material xlink:href="tx311.pdf" orientation="portrait" position="float"></supplementary-material><history><date date-type="received"><day>18</day><month>11</month><year>1997</year></date><date date-type="asap"><day>26</day><month>02</month><year>1998</year></date><date date-type="issue-pub"><day>20</day><month>04</month><year>1998</year></date></history><permissions><copyright-statement>Copyright © 1998 American Chemical Society</copyright-statement><copyright-year>1998</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The carcinogen ethylene dibromide (EDB) has been shown to cause
glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions,
in a variety of bacterial
and eukaryotic systems. The known DNA adducts
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH, and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
were individually placed at a site in a single
oligonucleotide. Polymerase extension studies were carried out
using <italic toggle="yes">Escherichia coli</italic>
polymerase I exo<sup>-</sup> (Klenow fragment, Kf<sup>-</sup>)
and polymerase II exo<sup>-</sup> (pol II<sup>-</sup>),
bacteriophage T7
polymerase exo<sup>-</sup>, and human immunodeficiency virus-1
reverse transcriptase in order to
characterize misincorporation events. Even though extension was
not as efficient as with the
nonadducted template, some fully extended primers were observed with
the template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
using all of these polymerases. dCTP was the most
preferred
nucleotide incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
by most of polymerases examined;
however, dTTP incorporation was observed opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
with pol II<sup>-</sup>.
Both
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
strongly blocked replication
by all polymerases. Only dATP and dGTP were incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH by both Kf<sup>-</sup> and pol II<sup>-</sup>.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-Guanyl)ethyl]GSH
was shown to strongly code for dATP
incorporation by Kf<sup>-</sup>. With pol II<sup>-</sup>,
dTTP was incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the
carcinogen EDB blocked
the polymerases and were capable of miscoding.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>tx970206m</meta-value></custom-meta></custom-meta-group></article-meta><notes id="tx970206mAF2"><label>†</label><p>
This research was supported in part by United
States Public
Health Service Grant R35 CA44353 and Grant P30 ES00267.
M.-S.
Kim was supported in part by a Merck predoctoral
fellowship.</p></notes></front><body><sec id="d7e257"><title>Introduction</title><p>Ethylene dibromide (EDB)<xref rid="tx970206mb00001" ref-type="bibr"></xref> has been
shown to induce
tumors in rodents at a number of sites (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00001" ref-type="bibr"></xref>−<xref rid="tx970206mb00002" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00004" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00005" ref-type="bibr"></xref></named-content></italic>).
The human
carcinogenicity of low-level EDB exposure is unknown,
but two workers died shortly following acute exposure
to EDB (<italic toggle="yes"><xref rid="tx970206mb00006" ref-type="bibr"></xref></italic>). Because of concern about toxicity
and
carcinogenicity, industrial use of EDB (soil fumigant,
scavenger in leaded gasoline) was considerably curtailed
in the 1980s (<italic toggle="yes"><xref rid="tx970206mb00007" ref-type="bibr"></xref></italic>).
</p><p>Bacteriophage M13mp18 was treated with
<italic toggle="yes">S</italic>-(2-chloroethyl)GSH, an analogue of the activated form of EDB,
and used to transfect <italic toggle="yes">Salmonella typhimurium</italic>;
DNA
sequence analysis of the <italic toggle="yes">lac Z</italic> α-complementation
mutants revealed that GC to AT transitions were dominant
(<italic toggle="yes"><xref rid="tx970206mb00008" ref-type="bibr"></xref></italic>). The same mutational changes (GC to AT
base
transitions) have also been observed in very different
biological systems such as <italic toggle="yes">Drosophila
melanogaster</italic>,
Chinese hamster ovary cells, and <italic toggle="yes">Escherichia coli
</italic>after
treatment with EDB, indicating that the biology related
to EDB adducts may be rather similar among species
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00009" ref-type="bibr"></xref>−<xref rid="tx970206mb00010" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00011" ref-type="bibr"></xref></named-content></italic>).
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-Guanyl)ethyl]GSH
was characterized as the
major DNA adduct derived from EDB, and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH,
and <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>1</sup>-adenyl)ethyl]GSH are minor adducts
(Chart <xref rid="tx970206mc00001"></xref>) (<italic toggle="yes">8</italic>,
<italic toggle="yes">12</italic>−<italic toggle="yes">15</italic>).
<fig id="tx970206mc00001" position="float" fig-type="chart" orientation="portrait"><label>1</label><caption><p>Structures of Guanyl Adducts Derived from EDB</p></caption><graphic xlink:href="tx970206mc00001.eps" position="float" orientation="portrait"></graphic></fig></p><p>Oligonucleotides containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
have been characterized by physicochemical methods
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00016" ref-type="bibr"></xref>−<xref rid="tx970206mb00017" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00018" ref-type="bibr"></xref></named-content></italic>). Base
pairing of
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
with
Cyt in the complementary sequence was shown to be
quite disrupted; however, the basic B-type helical structure was not perturbed (<italic toggle="yes"><xref rid="tx970206mb00016" ref-type="bibr"></xref></italic>). Comparative studies
of
oligonucleotides containing various <italic toggle="yes">N</italic><sup>7</sup>-guanyl
adducts
indicated some interactions between the GSH side chain
and the DNA bases (<italic toggle="yes"><xref rid="tx970206mb00017" ref-type="bibr"></xref></italic>). Dramatic variations of the
ratio
of <italic toggle="yes">S. typhimurium</italic> TA100 base-pair mutations to
bacterial DNA <italic toggle="yes">N</italic><sup>7</sup>-guanyl adducts were also observed
(<italic toggle="yes"><xref rid="tx970206mb00019" ref-type="bibr"></xref></italic>). The
frequency of <italic toggle="yes">O</italic><sup>6</sup>-alkylGua lesions in DNA has been
shown
to be strongly correlated with mutation induction in
different target genes in bacteria and cultured cells and
to be critical in tumorigenesis of many chemical carcinogens (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00020" ref-type="bibr"></xref>−<xref rid="tx970206mb00021" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00022" ref-type="bibr"></xref></named-content></italic>). GC to AT transition is the
predominant
type of mutation caused by <italic toggle="yes">O</italic><sup>6</sup>-alkylGua
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00023" ref-type="bibr"></xref>−<xref rid="tx970206mb00024" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00025" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00026" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00027" ref-type="bibr"></xref></named-content></italic>). Some
<italic toggle="yes">N</italic><sup>2</sup>-guanyl adducts have been shown to miscode
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00028" ref-type="bibr"></xref>, <xref rid="tx970206mb00029" ref-type="bibr"></xref></named-content></italic>).
</p><p>We developed methods for the synthesis of oligonucleotides containing the EDB-derived DNA adducts
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH,
and <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH at a single
site (<italic toggle="yes"><xref rid="tx970206mb00030" ref-type="bibr"></xref></italic>), in a sequence previously demonstrated to show mutations when
M13mp18 DNA was treated with
<italic toggle="yes">S</italic>-(2-chloroethyl)GSH,
an analogue of the activated form of EDB (<italic toggle="yes"><xref rid="tx970206mb00008" ref-type="bibr"></xref></italic>). In
this
study, the blocking and miscoding potentials of each Gua
adduct in the oligonucleotide were investigated using four
model DNA polymerases in order to characterize the
mechanism by which EDB causes mutations.
</p></sec><sec id="d7e478"><title>Experimental Procedures</title><p><bold>Chemicals.</bold> [γ-<sup>32</sup>P]dATP was
purchased from DuPont NEN
(Boston, MA). dNTPs were from Pharmacia (Piscataway,
NJ).
DNA sequencing systems (Maxam−Gilbert procedure)
were
from DuPont Biotechnology Systems (Boston, MA) and Sigma
Chemical Co. (St. Louis, MO).
</p><p><bold>Enzymes.</bold> T4 polynucleotide kinase was purchased
from
United States Biochemical (Cleveland, OH). DNA
polymerases
were purified by L. L. Furge, Department of Biochemistry,
from
<italic toggle="yes">E</italic>. <italic toggle="yes">coli</italic> using stock plasmids provided
(ε<sub>280</sub> values used in
parentheses): Kf<sup>-</sup> (Klenow fragment of <italic toggle="yes">E.
coli</italic> polymerase I,
exo<sup>-</sup>) (Dr. C. Joyce, Yale University, New Heaven, CT;
ε<sub>280</sub> =
6.32 × 10<sup>4</sup> M<sup>-1</sup>
cm<sup>-1</sup>), pol II<sup>-</sup> (<italic toggle="yes">E. coli
</italic>polymerase II exo<sup>-</sup>) (Prof.
M. F. Goodman, University of Southern California, Los
Angeles,
CA; ε<sub>280</sub> = 1.24 × 10<sup>5</sup>
M<sup>-1</sup> cm<sup>-1</sup>),
T7<sup>-</sup> (bacteriophage polymerase
T7 exo<sup>-</sup>; abbreviation T7<sup>-</sup> also refers to
thioredoxin mixture)
(ε<sub>280</sub> = 1.44 × 10<sup>5</sup>
M<sup>-1</sup> cm<sup>-1</sup>) and
thioredoxin (ε<sub>280</sub> = 1.37 ×
10<sup>4</sup>
M<sup>-1</sup> cm<sup>-1</sup>) (Prof. K.
A. Johnson, Pennsylvania State University,
University Park, PA), HIV RT (human immunodeficiency
virus-1 reverse transcriptase) (Dr. S. Hughes, Frederick
Cancer
Facility, Frederick, MD; ε<sub>280</sub> = 2.61 ×
10<sup>5</sup> M<sup>-1</sup>
cm<sup>-1</sup>) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00031" ref-type="bibr"></xref>,
<xref rid="tx970206mb00032" ref-type="bibr"></xref></named-content></italic>).
Concentrations of diluted solutions were estimated by
<italic toggle="yes">A</italic><sub>280</sub>
measurements using a modified Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). The ratio
of
T7 DNA polymerase to thioredoxin used in this study was
1:20.
</p><p><bold>Polymerase Assays. </bold><bold>General.</bold> The
synthesis and characterization of the three modified templates have been
described
(<italic toggle="yes"><xref rid="tx970206mb00030" ref-type="bibr"></xref></italic>). The 13-mer primer d(5‘-CTCGGTACCCTTG-3‘) (2
mM)
was 5‘-end-phosphorylated with <sup>32</sup>P using T4
polynucleotide
kinase and [γ-<sup>32</sup>P]dATP and purified on a Biospin
column (Bio-Rad, Hercules, CA). Template and primer (2:1 molar ratio)
were
annealed in a buffer containing 50 mM sodium MOPS
[3-(<italic toggle="yes">N</italic>-morpholino)propanesulfonic acid] (pH 7.0), bovine serum
albumin (50 μg mL<sup>-1</sup>), and 5 mM MgCl<sub>2</sub>
by incubating for 2 h at 16
°C. Concentrations of oligonucleotides were estimated
using
<italic toggle="yes">A</italic><sub>260</sub> measurements, with calculation of
extinction coefficients
as described (<italic toggle="yes"><xref rid="tx970206mb00033" ref-type="bibr"></xref></italic>): primer ε<sub>260</sub> = 8.54 ×
10<sup>4</sup> M<sup>-1</sup>
cm<sup>-1</sup>, template
ε<sub>260</sub> = 1.74 × 10<sup>5</sup>
M<sup>-1</sup> cm<sup>-1</sup>. The
extended products were
analyzed using a PhosphorImager system (model 400E, Molecular Dynamics, Sunnyvale, CA) and the manufacturer's software.
</p><p><bold>Primer Extension.</bold> Primer extension reactions were
performed by reacting primer/template pairs (100 nM) (Chart
<xref rid="tx970206mc00002"></xref>)
with a mixture of dNTPs (100 μM each) in 5 μL of 50 mM
sodium
MOPS buffer (pH 7.0) containing 8 mM MgCl<sub>2</sub>, 4 mM
dithiothreitol, and bovine serum albumin (2 μg
mL<sup>-1</sup>) with each of
three different concentrations of polymerases. These
reactions
were carried out for 1 h at 25 °C with all polymerases,
except
for pol II<sup>-</sup> which was incubated at 37 °C.<xref rid="tx970206mb00002" ref-type="bibr"></xref>
Reactions were
quenched by adding 5 μL of 10 mM EDTA in 90% formamide
(v/v), and the products were analyzed on 20% (w/v)
denaturing
polyacrylamide gels, prepared using Sequagel (National
Diagnostics, Atlanta, GA).
<fig id="tx970206mc00002" position="float" fig-type="chart" orientation="portrait"><label>2</label><caption><p>Oligonucleotides<italic toggle="yes"><sup>a</sup></italic><sup></sup></p><p><fn id="d7e693"><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> G* = Gua,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH, or
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH. The site
of first incorporation is indicated with an arrow.</p></fn></p></caption><graphic xlink:href="tx970206mc00002.eps" position="float" orientation="portrait"></graphic></fig></p><p><bold>One-Base Incorporation.</bold> Primers (13-mer) were
extended
using unmodified and adducted templates (100 nM) (Chart <xref rid="tx970206mc00002"></xref>)
in the presence of single dNTPs (100 μM). Preliminary
reactions
were performed at 25 °C for 1 h with all polymerases
(except
for pol II<sup>-</sup>, which was incubated at 37
°C),<xref rid="tx970206mb00002" ref-type="bibr"></xref> and the
concentrations of polymerase used were 200 nM (Kf<sup>-</sup>), 400 nM (pol
II<sup>-</sup>),
400 nM (T7), and 200 nM (HIV RT). Conditions were
adjusted
accordingly for steady-state kinetics (vide infra).
</p><p><bold>Steady-State Kinetics.</bold> Primer extension reactions
were
performed according to the general procedure described
elsewhere (<italic toggle="yes">31</italic>, <italic toggle="yes">32</italic>, <italic toggle="yes">35</italic>), using adducted
templates in the presence of
several different concentrations of single dNTPs as
indicated
in the table of the results. In all cases, extension was <20%
of
the substrate. The results were analyzed using a k•cat
computer program (Biometallics, Princeton, NJ).
</p><p><bold>Nucleotide Sequence Analysis.</bold> Nucleotide sequences
of
some of the primer extension products were analyzed as
described by Maxam and Gilbert (<italic toggle="yes"><xref rid="tx970206mb00036" ref-type="bibr"></xref></italic>), except for the
T-specific
reaction (<italic toggle="yes"><xref rid="tx970206mb00037" ref-type="bibr"></xref></italic>).
</p></sec><sec id="d7e774"><title>Results</title><p><bold>Polymerase Extension Assays.</bold> Primer
extension
reactions were performed by reacting primer/template
complexes with a mixture of all four dNTPs. All of
the
modified templates blocked extension to different degrees
(Figure <xref rid="tx970206mf00001"></xref>). Full-length extended
products were obtained
with templates containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
using Kf<sup>-</sup>, pol II<sup>-</sup>, T7<sup>-</sup>, and
HIV RT, even though
extension was not as efficient as with the control template. With Kf<sup>-</sup> and HIV RT, the one-base
extended
primer (14-mer) was observed as well as full-length
extended product, indicating that this adduct blocks
replication after addition of a single dNTP.
<fig id="tx970206mf00001" position="float" orientation="portrait"><label>1</label><caption><p>Extension of 13-mer primer in the presence of all four dNTPs when paired with modified 18-mers: (A) Kf<sup>-</sup>, (B) pol
II<sup>-</sup>,
(C) T7<sup>-</sup>, (D) HIV RT. The indicated concentrations of
polymerases were used, and other conditions are presented in the
Experimental
Procedures.</p></caption><graphic xlink:href="tx970206mf00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>Both
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH strongly blocked replication with Kf<sup>-</sup>.
One-base extended primers (14-mers) were found to be the
major products with both adducts. Nevertheless, a
small
amount of full-length extended product was observed
with the
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH-containing
oligonucleotide using Kf<sup>-</sup>. With pol II<sup>-</sup>, the
template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
was extended to yield a
ladder of all the possible products differing in length by
one base.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-Guanyl)ethyl]GSH
strongly blocked
replication with pol II<sup>-</sup>. With T7<sup>-</sup> and
HIV RT, the
primers were hardly extended with oligonucleotides
containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
or <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH and, as a result, could not be used
in
subsequent sequence analysis (vide infra).
</p><p><bold>Primer Extension Assays with Individual
dNTPs: Preliminary Studies.</bold> Extension studies
were
repeated with only each single nucleotide present at high
concentration and with prolonged incubation time, to
obtain qualitative information about misincorporation.
With Kf<sup>-</sup>, dCTP was the base preferentially
incorporated
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
although some incorporation of each dNTP could be detected under these
conditions of high dNTP concentration and extended
time.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-Guanyl)ethyl]GSH
showed strong mispairing with Kf<sup>-</sup>; both dATP and dGTP were
preferentially incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH.
Preferential incorporation of dATP opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH was observed with
Kf<sup>-</sup>.
</p><p>With pol II<sup>-</sup>, both dTTP and dCTP were
incorporated
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
as well as lesser
amounts of dGTP and dATP. dATP was the preferred
base incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH,
and both dTTP and dCTP were incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
using pol II<sup>-</sup>.
</p><p>With T7<sup>-</sup> and HIV RT, dCTP was incorporated
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH.
No incorporation was observed opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
or <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH with T7<sup>-</sup> or HIV
RT.
</p><p><bold>Nucleotide Sequence Analysis of Extended Primers.</bold> Extended products formed in the presence of all
four
dNTPs were analyzed (<italic toggle="yes"><xref rid="tx970206mb00036" ref-type="bibr"></xref></italic>). Only the reactions with
Kf<sup>-</sup>
and pol II<sup>-</sup> yielded sufficient material for analysis,
and
the results should be considered qualitative because of
the low signal and high background. Sequence analysis
of full-length primer-extended product from template
containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
showed that dCTP
was primarily incorporated opposite the adduct when
Kf<sup>-</sup>
was used (data not presented). In the 14-mer
(one-base
extended) product derived from the template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH,
both dATP and dGTP were
incorporated opposite the adduct (the “G + A” lane
band
was considerably more intense than the “G” lane band;
see Supporting Information). dATP was found to be
primarily incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH by analysis of the sequence of the 14-mer extended
by Kf<sup>-</sup>.
</p><p>Sequence analysis of fully extended primer from the
reaction of template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH with pol II<sup>-</sup> indicated that dATP was
incorporated
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
(data not presented).
</p><p>These qualitative results correspond with those from
the initial primer extension assays with individual
dNTPs.
</p><p><bold>Steady-State Kinetic Analysis of One-Base Incorporation.</bold> To obtain more quantitative information
about the tendency of DNA polymerases to misincorporate nucleotides opposite EDB-guanyl adducts, steady-state kinetic analyses were done under typical conditions
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00035" ref-type="bibr"></xref>, <xref rid="tx970206mb00038" ref-type="bibr"></xref></named-content></italic>) using both Kf<sup>-</sup> and pol
II<sup>-</sup> (Table <xref rid="tx970206mt00001"></xref>).
T7<sup>-</sup> and
HIV RT were not further examined due to previous
results (vide supra, lack of obvious incorporation of any
dNTPs except dCTP). All kinetic parameters
(<italic toggle="yes">K</italic><sub>m</sub> and
<italic toggle="yes">k</italic><sub>cat</sub>) were estimated
by nonlinear regression analysis of
the data. The relative efficiency of incorporation
was
determined from the ratio
<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>.
Misinsertion ratios
(also termed “misincorporation frequencies”) were
evaluated from the ratio of insertion frequency of wrong base
to right base,
[<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>(wrong)]/[<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>(right)],
opposite
specific adducts (<italic toggle="yes"><xref rid="tx970206mb00034" ref-type="bibr"></xref></italic>).
<table-wrap id="tx970206mt00001" position="float" orientation="portrait"><label>1</label><caption><p>Steady-State Kinetic Parameters for DNTP Incorporation</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="9"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:colspec colnum="8" colname="8"></oasis:colspec><oasis:colspec colnum="9" colname="9"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry namest="2" nameend="5">Kf<sup>-</sup></oasis:entry><oasis:entry namest="6" nameend="9">pol II<sup>-</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pairing</oasis:entry><oasis:entry colname="2"><italic toggle="yes">k</italic><sub>cat</sub>
(min<sup>-1</sup>)</oasis:entry><oasis:entry colname="3"><italic toggle="yes">K</italic><sub>m</sub>
(mM)</oasis:entry><oasis:entry colname="4"><italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub></oasis:entry><oasis:entry colname="5">misinsertion
ratio<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry colname="6"><italic toggle="yes">k</italic><sub>cat</sub>
(min<sup>-1</sup>)</oasis:entry><oasis:entry colname="7"><italic toggle="yes">K</italic><sub>m</sub>
(μM)</oasis:entry><oasis:entry colname="8"><italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub></oasis:entry><oasis:entry colname="9">misinsertion
ratio<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua:dCTP
</oasis:entry><oasis:entry colname="2">29<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">0.03
</oasis:entry><oasis:entry colname="4">970
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">21<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">0.11
</oasis:entry><oasis:entry colname="8">190
</oasis:entry><oasis:entry colname="9"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua:dATP
</oasis:entry><oasis:entry colname="2">0.71
</oasis:entry><oasis:entry colname="3">42
</oasis:entry><oasis:entry colname="4">0.017
</oasis:entry><oasis:entry colname="5">1.7 × 10<sup>-5</sup></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"><10<sup>-5</sup></oasis:entry><oasis:entry colname="9"><5 × 10<sup>-8</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua:dGTP
</oasis:entry><oasis:entry colname="2">0.45
</oasis:entry><oasis:entry colname="3">14
</oasis:entry><oasis:entry colname="4">0.032
</oasis:entry><oasis:entry colname="5">3.3 × 10<sup>-5</sup></oasis:entry><oasis:entry colname="6">0.072
</oasis:entry><oasis:entry colname="7">760
</oasis:entry><oasis:entry colname="8">9.4 × 10<sup>-5</sup></oasis:entry><oasis:entry colname="9">5 × 10<sup>-7</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua:dTTP
</oasis:entry><oasis:entry colname="2">0.57
</oasis:entry><oasis:entry colname="3">61
</oasis:entry><oasis:entry colname="4">0.0093
</oasis:entry><oasis:entry colname="5">1.0 × 10<sup>-5</sup></oasis:entry><oasis:entry colname="6">0.083
</oasis:entry><oasis:entry colname="7">770
</oasis:entry><oasis:entry colname="8">1.1 × 10<sup>-4</sup></oasis:entry><oasis:entry colname="9">6 × 10<sup>-7</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">N</italic><sup>7</sup>-Gua:dCTP
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">0.75<italic toggle="yes"><sup>d</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">0.24
</oasis:entry><oasis:entry colname="8">3.1
</oasis:entry><oasis:entry colname="9"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">N</italic><sup>7</sup>-Gua:dTTP
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">0.63<italic toggle="yes"><sup>e</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">340
</oasis:entry><oasis:entry colname="8">0.0019
</oasis:entry><oasis:entry colname="9">6 × 10<sup>-4</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">N</italic><sup>2</sup>-Gua:dCTP
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"><2 × 10<sup>-8</sup> <italic toggle="yes"><sup>f</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"><2 ×10<sup>-8</sup> <italic toggle="yes"><sup>g</sup></italic><sup></sup></oasis:entry><oasis:entry colname="9"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">N</italic><sup>2</sup>-Gua:dATP
</oasis:entry><oasis:entry colname="2">0.11<italic toggle="yes"><sup>h</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">80
</oasis:entry><oasis:entry colname="4">0.0014
</oasis:entry><oasis:entry colname="5">>10<sup>5</sup></oasis:entry><oasis:entry colname="6">0.043<italic toggle="yes"><sup>i</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">4400
</oasis:entry><oasis:entry colname="8">9.7 × 10<sup>-6</sup></oasis:entry><oasis:entry colname="9">>490
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">N</italic><sup>2</sup>Gua:dGTP
</oasis:entry><oasis:entry colname="2">0.40<italic toggle="yes"><sup>j</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">18
</oasis:entry><oasis:entry colname="4">0.022
</oasis:entry><oasis:entry colname="5">>10<sup>6</sup></oasis:entry><oasis:entry colname="6">0.0013<italic toggle="yes"><sup>k</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">440
</oasis:entry><oasis:entry colname="8">2.9 × 10<sup>-6</sup></oasis:entry><oasis:entry colname="9">>150
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">O</italic><sup>6</sup>-Gua:dCTP
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"><2 × 10<sup>-8</sup> <italic toggle="yes"><sup>l</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">1.8 × 10<sup>-4</sup> <italic toggle="yes"><sup>m</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">0.54
</oasis:entry><oasis:entry colname="8">3.3 × 10<sup>-4</sup></oasis:entry><oasis:entry colname="9"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">O</italic><sup>6</sup>-Gua:dATP
</oasis:entry><oasis:entry colname="2">0.027<italic toggle="yes"><sup>n</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">3.9
</oasis:entry><oasis:entry colname="4">0.007
</oasis:entry><oasis:entry colname="5">>10<sup>5</sup></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry><oasis:entry colname="9"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">O</italic><sup>6</sup>-Gua:dTTP
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">2.0 × 10<sup>-4</sup> <italic toggle="yes"><sup>o</sup></italic><sup></sup></oasis:entry><oasis:entry colname="7">4.4
</oasis:entry><oasis:entry colname="8">4.5 × 10<sup>-5</sup></oasis:entry><oasis:entry colname="9">0.13</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Misinsertion ratio =
(<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>)<sub>dNTP</sub>/(<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>)<sub>dCTP</sub>,
where dNTP ≠ dCTP (also termed misincorporation frequency)
(<italic toggle="yes"><xref rid="tx970206mb00034" ref-type="bibr"></xref></italic>). In cases where
the ratio <italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub> is
presented (as a limit) in the absence of <italic toggle="yes">k</italic><sub>cat</sub>
and <italic toggle="yes">K</italic><sub>m</sub>, <italic toggle="yes">k</italic><sub>cat</sub> was not
above the limit of detection and the limit at the highest
dNTP concentration is divided by that dNTP concentration to estimate a
limit for the ratio
<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>. In all cases the
[primer·template] was
100 nM. Some of the individual reaction conditions are indicated in the
footnotes.<italic toggle="yes"><sup>b</sup></italic><sup></sup> [Kf<sup>-</sup>] = 0.2
nM, [dCTP] = 0.01−5 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 1
min.<italic toggle="yes"><sup>c</sup></italic><sup></sup> [pol
II<sup>-</sup>] = 20 nM, [dCTP] = 0.01−50 <italic toggle="yes">m</italic>M,
<italic toggle="yes">t</italic> = 0.5 min.<italic toggle="yes"><sup>d</sup></italic><sup></sup> [pol
II<sup>-</sup>] = 40 nM, [dCTP] = 0.01−50 <italic toggle="yes">m</italic>M,
<italic toggle="yes">t</italic> = 6 min.<italic toggle="yes"><sup>e</sup></italic><sup></sup> [pol
II<sup>-</sup>] = 40 nM, [dTTP] =
0.5−1000 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 6
min.<italic toggle="yes"><sup>f</sup></italic><sup></sup> [Kf<sup>-</sup>] = 100 nM,
[dCTP] = 10 mM, <italic toggle="yes">t</italic> = 60
min.<italic toggle="yes"><sup>g</sup></italic><sup></sup> [pol II<sup>-</sup>] = 400
nM, [dCTP] = 10 mM, <italic toggle="yes">t</italic> = 60
min.<italic toggle="yes"><sup>h</sup></italic><sup></sup> [Kf<sup>-</sup>]
=
10 nM, [dATP] = 0.5−1000 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 10
min.<italic toggle="yes"><sup>i</sup></italic><sup></sup> [pol II<sup>-</sup>] = 40
nM, [dATP] = 0.005−10 mM, <italic toggle="yes">t</italic> = 30
min.<italic toggle="yes"><sup>j</sup></italic><sup></sup> [Kf<sup>-</sup>] = 10 nM,
[dGTP] = 0.5−1000
<italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 2
min.<italic toggle="yes"><sup>k</sup></italic><sup></sup> [pol II<sup>-</sup>] = 400
nM, [dGTP] = 0.005−10 mM, <italic toggle="yes">t</italic> = 20
min.<italic toggle="yes"><sup>l</sup></italic><sup></sup> [Kf<sup>-</sup>] = 100 nM,
[dCTP] = 10 mM, <italic toggle="yes">t</italic> = 60
min.<italic toggle="yes"><sup>m</sup></italic><sup></sup> [pol II<sup>-</sup>]
=
400 nM, [dCTP] = 0.5−1000 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 60
min.<italic toggle="yes"><sup>n</sup></italic><sup></sup> [Kf<sup>-</sup>] = 10 nM,
[dATP] = 0.5−1000 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 20
min.<italic toggle="yes"><sup>o</sup></italic><sup></sup> [pol II<sup>-</sup>] = 400
nM, [dTTP] =
0.5−1000 <italic toggle="yes">m</italic>M, <italic toggle="yes">t</italic> = 60 min.</p></table-wrap-foot></table-wrap></p><p>Misinsertion ratios were measured for insertion of
dATP, dGTP, and dTTP opposite G, relative to dCTP.
These ratios were on the order of 10<sup>-5</sup>
for Kf<sup>-</sup> and 10<sup>-7</sup>
for pol II<sup>-</sup> (Table <xref rid="tx970206mt00001"></xref>) and are considered in
subsequent
comparisons.
</p><p>With pol II<sup>-</sup>, dTTP incorporation was observed
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH,
as well as dCTP incorporation. The <italic toggle="yes">k</italic><sub>cat</sub> values for both dCTP
incorporation and
dTTP were similar; however, the <italic toggle="yes">K</italic><sub>m</sub> for the
incorporation
of dTTP was much higher than for dCTP incorporation.
Attempts with dATP and dGTP yielded much less
incorporation than for dTTP under these conditions, and
the results were not quantified. In a separate study
with
a commercial preparation of Kf<sup>-</sup>, no difference in
the
misinsertion ratios (for dTTP/dCTP) opposite Gua and
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
was detected (these results
are not included in Table <xref rid="tx970206mt00001"></xref> because of the different
preparation of enzyme used).
</p><p>Kf<sup>-</sup> and pol II<sup>-</sup> incorporated both dATP and
dGTP
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH.
There was no detectable dCTP incorporation opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH with either DNA polymerase. The
estimates
of <italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub> are based on
limits of detection at high
substrate concentrations. The apparent misinsertion
ratios, then, are very high for both dATP and dGTP.
With
Kf<sup>-</sup> the
<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub> ratio was 15, in
favor of dGTP incorporation over dATP. However,
<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub> was 3 times
greater
for dATP incorporation over dGTP with pol
II<sup>-</sup>.
</p><p>Kf<sup>-</sup> incorporated dATP opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH, and there was no detectable dCTP base incorporation. dTTP incorporation was observed opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH, as well as dCTP, using pol
II<sup>-</sup>. <italic toggle="yes">k</italic><sub>cat</sub>
for the incorporation of both bases was similar; however,
the <italic toggle="yes">K</italic><sub>m</sub> for dTTP incorporation was 10 times
greater than
for dCTP incorporation with pol II<sup>-</sup>, yielding a
misincorporation ratio of 0.13.
</p></sec><sec id="d7e2021"><title>Discussion</title><p>All three of the guanyl-ethylene-GSH adducts
(<italic toggle="yes">N</italic><sup>7</sup>, <italic toggle="yes">N</italic><sup>2</sup>,
and <italic toggle="yes">O</italic><sup>6</sup>) were shown to block replication and
miscode with
at least one polymerase. The extents of replication
blockage and miscoding specificity were dependent on
both the adduct structure and polymerase used.
</p><p>Chemical lability has hindered the study of biological
properties of <italic toggle="yes">N</italic><sup>7</sup>-alkylGua derivatives, i.e.,
instability of
the glycosidic bond and the tendency of the charged
imidazole ring to open. In general,
<italic toggle="yes">N</italic><sup>7</sup>-alkylGua derivatives have been considered not to be miscoding lesions
in most of the literature. No <italic toggle="yes">N</italic><sup>7</sup>-alkylGua
adducts have
been definitively shown to miscode except one, that
derived from aflatoxin B<sub>1</sub> <italic toggle="yes">exo</italic>-8,9-epoxide
(<italic toggle="yes"><xref rid="tx970206mb00039" ref-type="bibr"></xref></italic>). We found
that
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
blocked replication and
caused a low level of miscoding. Extension of the
primer
annealed to the adducted template was not as efficient
as nonadducted template; however, some fully extended
primers were observed with all polymerases. dCTP was
the most preferred nucleotide incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH with
Kf<sup>-</sup>, T7<sup>-</sup>, and HIV RT; however, dTTP incorporation was detected opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH with pol II<sup>-</sup> (Table <xref rid="tx970206mt00001"></xref>).
The efficiency
of insertion (<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>)
for dCTP opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH decreased dramatically compared to Gua
(from 190 to 3.1), and the misinsertion ratio for dTTP
was 6 × 10<sup>-4</sup> (i.e., 0.06%). DNA
sequence analysis
indicated that dCTP was predominantly incorporated
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
in the primer, which
was fully extended with Kf<sup>-</sup> and pol II<sup>-</sup>.
However,
sequence analysis would not have been expected to detect
the presence of T, with such a low misincorporation
frequency. The misincorporation ratio of 6 ×
10<sup>-4</sup> may
be compared with the values seen with the oligonucleotide containing Gua at this site, which are 3 orders of
magnitude lower (Table <xref rid="tx970206mt00001"></xref>). This comparison argues
that
the misincorporation event is significant. However,
an
adduct level of ∼1 modification/10<sup>3</sup> bases of DNA
would
be required to double the overall extent of
misincorporation seen in the absence of the adduct.
</p><p><italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-Guanyl)ethyl]GSH
was a block to replication
and miscoded. With Kf<sup>-</sup> a strong replication block
was
observed after one base was incorporated; however, pol
II<sup>-</sup> incorporated one base beyond the adduct (Figure
<xref rid="tx970206mf00001"></xref>B).
dATP and dGTP were readily incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH (Table <xref rid="tx970206mt00001"></xref>). A
small amount of full-length product was detectable using the template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
and Kf<sup>-</sup>. Attempts to
analyze the DNA sequence of this product were not very
successful, because the amount of product was quite low.
With T7<sup>-</sup> and HIV RT, the primer paired with
the
template containing
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH
was hardly
extended.
</p><p><italic toggle="yes">O</italic><sup>6</sup>-AlkylGua derivatives are generally considered
to be
important mutagenic lesions, critical in tumorigenesis of
many chemical carcinogens, and cytotoxic
(<italic toggle="yes">20</italic>−<italic toggle="yes">22</italic>, <italic toggle="yes">40</italic>−<italic toggle="yes">42</italic>). dTTP is preferentially incorporated opposite
<italic toggle="yes">O</italic><sup>6</sup>-methylGua, causing GC to AT transitions (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00023" ref-type="bibr"></xref>−<xref rid="tx970206mb00024" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00025" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00026" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="tx970206mb00027" ref-type="bibr"></xref></named-content></italic>).
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-Guanyl)ethyl]GSH strongly blocked
replication with
all polymerases. dATP was primarily incorporated opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH,
and some incorporation of dGTP was observed with Kf<sup>-</sup>. The
misinsertion
ratio for dATP opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
was
>10<sup>5</sup>. dTTP incorporation and dCTP incorporation
opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
were both detectable
with pol II<sup>-</sup>. The misinsertion ratio for dTTP was
0.13,
which was lower than that reported for dTTP opposite
<italic toggle="yes">O</italic><sup>6</sup>-methylGua with Kf<sup>-</sup> in a
different sequence (1−2.5)
(<italic toggle="yes"><xref rid="tx970206mb00027" ref-type="bibr"></xref></italic>).
</p><p>In considering the results of the polymerase kinetic
experiments, comparisons can be made not only of the
misinsertion ratios but also of the enzyme efficiencies
(<italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>) for each
pairing. With regard to pairing of Gua
and its derivatives with dTTP by pol II<sup>-</sup>, the
efficiency
is ∼17-fold better for
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
than
Gua but the efficiencies for
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
and Gua are similar (Table <xref rid="tx970206mt00001"></xref>). With regard to
insertion
of dATP and dGTP, the efficiencies are less with both
the N<sup>2</sup>- and O<sup>6</sup>-adducts than with Gua for both
Kf<sup>-</sup> and
pol II<sup>-</sup>. The favorable misinsertion ratios are
driven not
by favorable pairing of dATP and dGTP with the adducts
but by the very disfavored incorporation of dCTP opposite
these adducts. The molecular basis of this disfavored
incorporation of dCTP (as much as 10<sup>11</sup>) may be
related
to the difficulty in developing canonical Watson−Crick
base pairs with dCTP due to the presence of bulky
<italic toggle="yes">N</italic><sup>2</sup>-
and <italic toggle="yes">O</italic><sup>6</sup>-guanyl adducts. Alternative pairing
modes (e.g.,
wobble) may be nearly as feasible for these derivatives
as for Gua itself. Exactly why the N<sup>7</sup>-adduct is a
block
(Table <xref rid="tx970206mt00001"></xref>, Figure <xref rid="tx970206mf00001"></xref>) is not clear, since the base modification is not in the base-pairing region and only changed
the ΔΔ<italic toggle="yes">G</italic>° for binding to a complemetary C by 1.4
kcal
mol<sup>-1</sup> (<italic toggle="yes"><xref rid="tx970206mb00018" ref-type="bibr"></xref></italic>). However, the GSH
moiety does present
considerable bulk in the major groove [when the DNA is
double-stranded (<italic toggle="yes"><xref rid="tx970206mb00016" ref-type="bibr"></xref></italic>)] and may interact directly with
the
polymerase.
</p><p>Abril et al. (<italic toggle="yes"><xref rid="tx970206mb00043" ref-type="bibr"></xref></italic>) reported decreased mutations
caused
by EDB in bacterial strains devoid of
<italic toggle="yes">O</italic><sup>6</sup>-alkyltransferases. The basis of this finding is not clear, because
if
the <italic toggle="yes">O</italic><sup>6</sup>-alkylGua lesion is mutagenic, the
transferases
might repair this lesion and lower the mutation rate.
Possible speculation on this finding is that a repair
enzyme might bind to
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
without
repairing it, and mutation could somehow be enhanced
by a complex of DNA lesion and repair enzyme. However, nucleotide excision repair does seem to be involved
and lowers the mutation frequency in experiments in
which DNA is modified with the half-mustard (2-chloroethyl)GSH (<italic toggle="yes"><xref rid="tx970206mb00008" ref-type="bibr"></xref></italic>). It is not known whether
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH is a substrate foror an inhibitor
of<italic toggle="yes">O</italic><sup>6</sup>-alkyltransferase. There was no detectable
incorporation
of any base opposite
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH
or <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-guanyl)ethyl]GSH with
T7<sup>-</sup> or HIV RT (Table <xref rid="tx970206mt00001"></xref>), both
of which are replicative polymerases, indicating that the
fates of replication of EDB-guanyl adducts are highly
dependent on DNA polymerases.
</p><p>In conclusion, the miscoding potentials of three known
EDB-guanyl adducts were investigated.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-Guanyl)ethyl]GSH was found to be a replication block
and
miscoded to insert dTTP, with pol II<sup>-</sup>, at a low
frequency.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>2</sup>-Guanyl)ethyl]GSH
was shown to be capable of
blocking polymerization and miscoding, with dATP and
dGTP being the most preferred bases incorporated opposite this adduct.
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-Guanyl)ethyl]GSH
was also
found to be a replication block and to code for dATP and
dGTP incorporation with Kf<sup>-</sup> and for dTTP
incorporation
with pol II<sup>-</sup>. In considering the GC to AT
transition
observed in many biological systems, the expected pairing
of a Gua adduct with dTTP was observed only with pol
II<sup>-</sup> and two adducts,
<italic toggle="yes">S</italic>-[2-(<italic toggle="yes">N</italic><sup>7</sup>-guanyl)ethyl]GSH
and <italic toggle="yes">S</italic>-[2-(<italic toggle="yes">O</italic><sup>6</sup>-guanyl)ethyl]GSH (Table <xref rid="tx970206mt00001"></xref>).
The O<sup>6</sup>-adduct was 2
orders of magnitude more likely to miscode than the
N<sup>7</sup>-adduct, but the N<sup>7</sup>-adduct is present at levels at least
2
orders of magnitude higher than the O<sup>6</sup>-adduct
(<italic toggle="yes"><xref rid="tx970206mb00008" ref-type="bibr"></xref></italic>). A
quantitative assessment of the ability of pol II<sup>-</sup> to
extend
the two adducts has not been made. The in vitro assay
system we used in this study is not necessarily representative of the prokaryotic cellular replication system,
in which DNA polymerase III is the major replicative
enzyme [prior work suggests that the SOS system is not
obligatory for mutagenesis in bacteria (<italic toggle="yes"><xref rid="tx970206mb00019" ref-type="bibr"></xref></italic>)]. This
study
has provided useful information about how these adducts
can behave with DNA polymerases, even though the
information about which guanyl adduct is primarily
responsible for the dominant GC to AT transitions seen
in vivo is still not complete.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Dr. S. Langouët for
valuable suggestions and L. L. Furge for purification of
the polymerases used in this study and for comments on
the manuscript.
</p></ack><notes notes-type="si"><sec id="d7e2517"><title><ext-link xlink:href="/doi/suppl/10.1021%2Ftx970206m">Supporting Information Available</ext-link></title><p>Figure showing
nucleotide sequence analysis of extended primers (1 page).
Ordering information is given on any current masthead
page.
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Toxicol.</italic>).</comment></mixed-citation></ref><ref id="tx970206mn00002"><mixed-citation><comment>The 37 °C temperature was used with pol II<sup>-</sup> because of historical reasons and for making comparisons with the literature; the other polymerases have been used historically at 25 °C (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="tx970206mb00031" ref-type="bibr"></xref>, <xref rid="tx970206mb00034" ref-type="bibr"></xref></named-content></italic>).</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†</title></titleInfo><titleInfo contentType="CDATA"><title>Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†</title></titleInfo><name type="personal"><namePart type="family">KIM</namePart><namePart type="given">Mi-Sook</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</affiliation><affiliation> Current address: Merck & Company, P.O. Box 2000,126 EastLincoln Ave., Rahway, NJ 07065-0900.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">GUENGERICH</namePart><namePart type="given">F. Peter</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232-0146</affiliation><affiliation> Address correspondence to this author. Tel: (615) 322-2261.Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-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>American Chemical Society</publisher><dateCreated encoding="w3cdtf">1998-02-26</dateCreated><dateIssued encoding="w3cdtf">1998-04-20</dateIssued><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><note type="footnote" ID="tx970206mAF2"> This research was supported in part by United States Public Health Service Grant R35 CA44353 and Grant P30 ES00267. M.-S. Kim was supported in part by a Merck predoctoral fellowship.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.</abstract><note type="footnote" ID="tx970206mAF2"> This research was supported in part by United States Public Health Service Grant R35 CA44353 and Grant P30 ES00267. M.-S. Kim was supported in part by a Merck predoctoral fellowship.</note><relatedItem type="host"><titleInfo><title>Chemical Research in Toxicology</title></titleInfo><titleInfo type="abbreviated"><title>Chem. Res. 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The standard abbreviations for nucleic acid bases are defined in the current Instructions to Authors (see the January issue ofChem. Res. Toxicol.).</title></titleInfo><note type="content-in-line">Abbreviations: EDB, ethylene dibromide; MOPS, 3-(N-morpholino)propanesulfonic acid; Kf-, Klenow fragment (of Escherichia coli polymerase I) exo-; pol II-, polymerase II exo-; T7-, bacteriophage polymerase T7 exo- (thioredoxin mixture); HIV RT, human immunodeficiency virus-1 reverse transcriptase. The standard abbreviations for nucleic acid bases are defined in the current Instructions to Authors (see the January issue of Chem. Res. Toxicol.).</note></relatedItem><relatedItem type="references" ID="tx970206mn00002" displayLabel="bibtx970206mn00002"><titleInfo><title>The 37 °C temperature was used with pol II-because of historical reasons and for making comparisons with the literature; the other polymerases have been used historically at 25 °C (,).</title></titleInfo><note type="content-in-line">The 37 °C temperature was used with pol II- because of historical reasons and for making comparisons with the literature; the other polymerases have been used historically at 25 °C (, ).</note></relatedItem><identifier type="istex">B587D6D00D21F938208F695EFBADD5C3568FA782</identifier><identifier type="ark">ark:/67375/TPS-QTD464B6-M</identifier><identifier type="DOI">10.1021/tx970206m</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 1998 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/record.json</uri></json:item></metadata><annexes><json:item><extension>eps</extension><original>true</original><mimetype>image/x-eps</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-QTD464B6-M/annexes.eps</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/B587D6D00D21F938208F695EFBADD5C3568FA782/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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|texte= Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione†
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