Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†
Identifieur interne : 001279 ( Istex/Corpus ); précédent : 001278; suivant : 001280Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†
Auteurs : Sophie Langouët ; Adrienne N. Mican ; Michael Müller ; Stephen P. Fink ; Lawrence J. Marnett ; Steven A. Muhle ; F. Peter GuengerichSource :
- Biochemistry [ 0006-2960 ] ; 1998.
Abstract
A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1,N2-Ethenoguanine (1,N2-ε-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G→A, 0.74% G→T, and 0.09% G→C changes found in uvrA- bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-ε-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007−0.19% for G→A, C, or T changes. The saturated etheno ring derivative of 1,N2-ε-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G→A and G→T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-ε-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.
Url:
DOI: 10.1021/bi972327r
Links to Exploration step
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†</title><author><name sortKey="Langouet, Sophie" sort="Langouet, Sophie" uniqKey="Langouet S" first="Sophie" last="Langouët">Sophie Langouët</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: INSERM U456, Faculté desSciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,35043Rennes cedex, France.</mods:affiliation></affiliation></author><author><name sortKey="Mican, Adrienne N" sort="Mican, Adrienne N" uniqKey="Mican A" first="Adrienne N." last="Mican">Adrienne N. Mican</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Muller, Michael" sort="Muller, Michael" uniqKey="Muller M" first="Michael" last="Müller">Michael Müller</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Abteilung Arbeits- undSozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</mods:affiliation></affiliation></author><author><name sortKey="Fink, Stephen P" sort="Fink, Stephen P" uniqKey="Fink S" first="Stephen P." last="Fink">Stephen P. Fink</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Marnett, Lawrence J" sort="Marnett, Lawrence J" uniqKey="Marnett L" first="Lawrence J." last="Marnett">Lawrence J. Marnett</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Muhle, Steven A" sort="Muhle, Steven A" uniqKey="Muhle S" first="Steven A." last="Muhle">Steven A. Muhle</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</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 of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Address correspondence to this author: Department ofBiochemistry, Vanderbilt University School of Medicine,638Medical Research Building I, Nashville, TN 37232-0146.Telephone: (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:004E2BD6D48F49FB7F81EBCFA16DC6B6631F8A9F</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1021/bi972327r</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-P1V0PCZ4-2/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001279</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001279</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine
Derivatives by <hi rend="italic">Escherichia c</hi><hi rend="italic">oli</hi> Polymerases in Vitro and in Vivo:
1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine, and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine<ref type="bib" target="#bi972327rAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Langouet, Sophie" sort="Langouet, Sophie" uniqKey="Langouet S" first="Sophie" last="Langouët">Sophie Langouët</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: INSERM U456, Faculté desSciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,35043Rennes cedex, France.</mods:affiliation></affiliation></author><author><name sortKey="Mican, Adrienne N" sort="Mican, Adrienne N" uniqKey="Mican A" first="Adrienne N." last="Mican">Adrienne N. Mican</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Muller, Michael" sort="Muller, Michael" uniqKey="Muller M" first="Michael" last="Müller">Michael Müller</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Abteilung Arbeits- undSozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</mods:affiliation></affiliation></author><author><name sortKey="Fink, Stephen P" sort="Fink, Stephen P" uniqKey="Fink S" first="Stephen P." last="Fink">Stephen P. Fink</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Marnett, Lawrence J" sort="Marnett, Lawrence J" uniqKey="Marnett L" first="Lawrence J." last="Marnett">Lawrence J. Marnett</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation></author><author><name sortKey="Muhle, Steven A" sort="Muhle, Steven A" uniqKey="Muhle S" first="Steven A." last="Muhle">Steven A. Muhle</name><affiliation><mods:affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</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 of Medicine,Nashville, Tennessee 37232-0146</mods:affiliation></affiliation><affiliation><mods:affiliation> Address correspondence to this author: Department ofBiochemistry, Vanderbilt University School of Medicine,638Medical Research Building I, Nashville, TN 37232-0146.Telephone: (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">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1998</date><date type="published">1998</date><biblScope unit="vol">37</biblScope><biblScope unit="issue">15</biblScope><biblScope unit="page" from="5184">5184</biblScope><biblScope unit="page" to="5193">5193</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1,N2-Ethenoguanine (1,N2-ε-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G→A, 0.74% G→T, and 0.09% G→C changes found in uvrA- bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-ε-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007−0.19% for G→A, C, or T changes. The saturated etheno ring derivative of 1,N2-ε-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G→A and G→T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-ε-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>ethanogua</json:string><json:string>adduct</json:string><json:string>mutation</json:string><json:string>polymerase</json:string><json:string>coli</json:string><json:string>exocyclic</json:string><json:string>biochemistry</json:string><json:string>oligonucleotides</json:string><json:string>chem</json:string><json:string>misincorporation</json:string><json:string>dctp</json:string><json:string>mutagenesis</json:string><json:string>marnett</json:string><json:string>toxicol</json:string><json:string>mutagenic</json:string><json:string>primer</json:string><json:string>dntps</json:string><json:string>uvra</json:string><json:string>dntp</json:string><json:string>hplc</json:string><json:string>datp</json:string><json:string>dgtp</json:string><json:string>kcat</json:string><json:string>dttp</json:string><json:string>ethanodguo</json:string><json:string>guengerich</json:string><json:string>mutation frequency</json:string><json:string>derivative</json:string><json:string>m1gua</json:string><json:string>blockage</json:string><json:string>reaction time</json:string><json:string>total mutation frequency</json:string><json:string>replication</json:string><json:string>ethano ring</json:string><json:string>coli strain</json:string><json:string>kcat value</json:string><json:string>assay</json:string><json:string>coli polymerase</json:string><json:string>adduct site</json:string><json:string>dntps opposite ethanogua</json:string><json:string>exocyclic ring</json:string><json:string>dntp concentration</json:string><json:string>nucleotide excision repair</json:string><json:string>more mutagenic</json:string><json:string>incorporation</json:string><json:string>nucleotide</json:string></teeft></keywords><author><json:item><name>LANGOUëT Sophie</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</json:string><json:string>Current address: INSERM U456, Faculté desSciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,35043Rennes cedex, France.</json:string></affiliations></json:item><json:item><name>MICAN Adrienne N.</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</json:string></affiliations></json:item><json:item><name>MüLLER Michael</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</json:string><json:string>Current address: Abteilung Arbeits- undSozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</json:string></affiliations></json:item><json:item><name>FINK Stephen P.</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</json:string></affiliations></json:item><json:item><name>MARNETT Lawrence J.</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</json:string></affiliations></json:item><json:item><name>MUHLE Steven A.</name><affiliations><json:string>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</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 of Medicine,Nashville, Tennessee 37232-0146</json:string><json:string>Address correspondence to this author: Department ofBiochemistry, Vanderbilt University School of Medicine,638Medical Research Building I, Nashville, TN 37232-0146.Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-P1V0PCZ4-2</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1,N2-Ethenoguanine (1,N2-ε-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G→A, 0.74% G→T, and 0.09% G→C changes found in uvrA- bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-ε-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007−0.19% for G→A, C, or T changes. The saturated etheno ring derivative of 1,N2-ε-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G→A and G→T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-ε-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.</abstract><qualityIndicators><score>10</score><pdfWordCount>7039</pdfWordCount><pdfCharCount>42562</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>10</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>704</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>297</abstractWordCount><abstractCharCount>1976</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>37</volume><issue>15</issue><pages><first>5184</first><last>5193</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-P1V0PCZ4-2</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</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>10.1021/bi972327r</json:string></doi><id>004E2BD6D48F49FB7F81EBCFA16DC6B6631F8A9F</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-P1V0PCZ4-2/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-P1V0PCZ4-2/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-P1V0PCZ4-2/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-P1V0PCZ4-2/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine
Derivatives by <hi rend="italic">Escherichia c</hi><hi rend="italic">oli</hi> Polymerases in Vitro and in Vivo:
1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine, and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine<ref type="bib" target="#bi972327rAF2"><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">Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine
Derivatives by <hi rend="italic">Escherichia c</hi><hi rend="italic">oli</hi> Polymerases in Vitro and in Vivo:
1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine, and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine<ref type="bib" target="#bi972327rAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Langouët</surname><forename type="first">Sophie</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="bi972327rAF3"><ref>‡</ref><p>
Current address: INSERM U456, Faculté des
Sciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,
35043
Rennes cedex, France.</p></note></author><author xml:id="author-0001"><persName><surname>Mican</surname><forename type="first">Adrienne N.</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></author><author xml:id="author-0002"><persName><surname>Müller</surname><forename type="first">Michael</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="bi972327rAF4"><ref>§</ref><p>
Current address: Abteilung Arbeits- und
Sozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073
Göttingen,
Germany.</p></note></author><author xml:id="author-0003"><persName><surname>Fink</surname><forename type="first">Stephen P.</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></author><author xml:id="author-0004"><persName><surname>Marnett</surname><forename type="first">Lawrence J.</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></author><author xml:id="author-0005"><persName><surname>Muhle</surname><forename type="first">Steven A.</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></author><author xml:id="author-0006" 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: Department of Biochemistry, Vanderbilt University School of Medicine, 638 Medical Research Building I, Nashville, TN 37232-0146. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@ toxicology.mc.vanderbilt.edu.</affiliation></author><idno type="istex">004E2BD6D48F49FB7F81EBCFA16DC6B6631F8A9F</idno><idno type="ark">ark:/67375/TPS-P1V0PCZ4-2</idno><idno type="DOI">10.1021/bi972327r</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1998</date><date type="published">1998</date><biblScope unit="vol">37</biblScope><biblScope unit="issue">15</biblScope><biblScope unit="page" from="5184">5184</biblScope><biblScope unit="page" to="5193">5193</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>A variety of exocyclic modified bases have been shown to be formed
in DNA from various
procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan)
and are also found in untreated
animals and humans, presumably arising as a result of lipid
peroxidation. 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine
(1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua), a product known to be formed from several 2-carbon electrophiles,
was placed in a known site
(6256) in bacteriophage M13MB19 and mutations were analyzed in
<hi rend="italic">Escherichia coli,</hi> with 2.05% G→A,
0.74% G→T, and 0.09% G→C changes found in uvrA<hi rend="superscript">-</hi>
bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine (HO-ethanoGua), formally
the hydrated derivative of 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua, is a
stable
DNA product also derived from vinyl halides. When this base was
placed in the same context, the mutation
rate was 0.007−0.19% for G→A, C, or T changes. The saturated
etheno ring derivative of
1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua,
5,6,7,9-tetrahydro-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine
(ethanoGua) produced G→A and G→T mutations (0.71%
each). All mutants were SOS-dependent and were attenuated by uvrA
activity in <hi rend="italic">E. coli</hi>. In vitro studies
with four polymerases showed strong blocks to addition beyond the
adduct site in the order ethanoGua
> HO-ethanoGua > 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua. Both
<hi rend="italic">E. coli</hi> polymerases (pol) I exo<hi rend="superscript">-</hi> and II
exo<hi rend="superscript">-</hi> and bacteriophage
pol T7 exo<hi rend="superscript">-</hi> showed extensive misincorporation opposite
ethanoGua in vitro, with pol I exo<hi rend="superscript">-</hi>
incorporating
G and T, pol II exo<hi rend="superscript">-</hi> incorporating A, and pol T7
exo<hi rend="superscript">-</hi> incorporating A and G. All modified
bases
reduced the use of the minus strand bearing the modified guanine in
<hi rend="italic">E. coli</hi> cells. It is of interest that
even though the normal base pairing site of guanine is completely
blocked, all of the five-membered ring
derivatives incorporate the normal base (C) in >80% of the
replication events in <hi rend="italic">E. coli</hi>. Major
differences
in blockage and misincorporation are seen due to what might appear to
be relatively modest structural
differences, and polymerases can differ dramatically in their
selectivities.
</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">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi972327r</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine
Derivatives by <italic toggle="yes">Escherichia c</italic><italic toggle="yes">oli</italic> Polymerases in Vitro and in Vivo:
1,<italic toggle="yes">N</italic><sup>2</sup>-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine, and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine<xref rid="bi972327rAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Langouët</surname><given-names>Sophie</given-names></name><xref rid="bi972327rAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Mican</surname><given-names>Adrienne N.</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Müller</surname><given-names>Michael</given-names></name><xref rid="bi972327rAF4"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Fink</surname><given-names>Stephen P.</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Marnett</surname><given-names>Lawrence J.</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Muhle</surname><given-names>Steven A.</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Guengerich</surname><given-names>F. Peter</given-names></name><xref rid="bi972327rAF1">*</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="bi972327rAF3"><label>‡</label><p>
Current address: INSERM U456, Faculté des
Sciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,
35043
Rennes cedex, France.</p></fn><fn id="bi972327rAF4"><label>§</label><p>
Current address: Abteilung Arbeits- und
Sozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073
Göttingen,
Germany.</p></fn><corresp id="bi972327rAF1">
Address correspondence to this author: Department of
Biochemistry, Vanderbilt University School of Medicine,
638
Medical Research Building I, Nashville, TN 37232-0146.
Telephone: (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>03</month><year>1998</year></pub-date><pub-date pub-type="ppub"><day>14</day><month>04</month><year>1998</year></pub-date><volume>37</volume><issue>15</issue><fpage>5184</fpage><lpage>5193</lpage><history><date date-type="received"><day>18</day><month>09</month><year>1997</year></date><date date-type="rev-recd"><day>15</day><month>01</month><year>1998</year></date><date date-type="asap"><day>26</day><month>03</month><year>1998</year></date><date date-type="issue-pub"><day>14</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>A variety of exocyclic modified bases have been shown to be formed
in DNA from various
procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan)
and are also found in untreated
animals and humans, presumably arising as a result of lipid
peroxidation. 1,<italic toggle="yes">N</italic><sup>2</sup>-Ethenoguanine
(1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua), a product known to be formed from several 2-carbon electrophiles,
was placed in a known site
(6256) in bacteriophage M13MB19 and mutations were analyzed in
<italic toggle="yes">Escherichia coli,</italic> with 2.05% G→A,
0.74% G→T, and 0.09% G→C changes found in uvrA<sup>-</sup>
bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine (HO-ethanoGua), formally
the hydrated derivative of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, is a
stable
DNA product also derived from vinyl halides. When this base was
placed in the same context, the mutation
rate was 0.007−0.19% for G→A, C, or T changes. The saturated
etheno ring derivative of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua,
5,6,7,9-tetrahydro-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine
(ethanoGua) produced G→A and G→T mutations (0.71%
each). All mutants were SOS-dependent and were attenuated by uvrA
activity in <italic toggle="yes">E. coli</italic>. In vitro studies
with four polymerases showed strong blocks to addition beyond the
adduct site in the order ethanoGua
> HO-ethanoGua > 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua. Both
<italic toggle="yes">E. coli</italic> polymerases (pol) I exo<sup>-</sup> and II
exo<sup>-</sup> and bacteriophage
pol T7 exo<sup>-</sup> showed extensive misincorporation opposite
ethanoGua in vitro, with pol I exo<sup>-</sup>
incorporating
G and T, pol II exo<sup>-</sup> incorporating A, and pol T7
exo<sup>-</sup> incorporating A and G. All modified
bases
reduced the use of the minus strand bearing the modified guanine in
<italic toggle="yes">E. coli</italic> cells. It is of interest that
even though the normal base pairing site of guanine is completely
blocked, all of the five-membered ring
derivatives incorporate the normal base (C) in >80% of the
replication events in <italic toggle="yes">E. coli</italic>. Major
differences
in blockage and misincorporation are seen due to what might appear to
be relatively modest structural
differences, and polymerases can differ dramatically in their
selectivities.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi972327r</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi972327rAF2"><label>†</label><p>
This research was supported in part by United
States Public Health
Service Grants R35 CA44353 (F.P.G.), R35 CA47479 (L.J.M.),
P30
ES00267 (F.P.G., L.J.M.), T32 ES07028 (A.N.M.), and T32
GM07347
(S.A.M.) and a fellowship from the Deutsche
Forschungsgemeinschaft
(M.M.).</p></notes></front><body><sec id="d7e260"><title></title><p>Modification of nucleic acids with bifunctional electrophiles results in the formation of a number of different
base
derivatives that contain an exocyclic five- or
six-membered
ring (<italic toggle="yes">1</italic><italic toggle="yes">−</italic><italic toggle="yes">4</italic>). Some similar
compounds have been identified
in tRNAs (<italic toggle="yes">5</italic>). In DNA, most of the chemicals that
form
such derivatives are known mutagens and carcinogens, and
there is considerable interest in the hypothesis that the
exocyclic bases are major contributors to the mutagenicity
of these chemicals (<italic toggle="yes">3</italic>). In support of this view, the
five-membered exocyclic derivatives
<italic toggle="yes">N</italic><sup>2</sup>,3-ε-Gua,<xref rid="bi972327rX00001" ref-type="bibr"></xref>
1,<italic toggle="yes">N</italic><sup>6</sup>-ε-Ade,
and 3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyd (Scheme
<xref rid="bi972327rh00001"></xref>) have all been shown to
produce
some mutations when examined in various systems, although
direct comparisons have not been made
(<italic toggle="yes">6</italic><italic toggle="yes">−</italic><italic toggle="yes">10</italic>). The six-
membered ring derivatives
M<sub>1</sub>Gua and propanoGua (Scheme
<xref rid="bi972327rh00002"></xref>) have been previously considered
for mutagenicity in one
of our laboratories and elsewhere and shown to be
miscoding
in vitro and in vivo (<italic toggle="yes">11</italic>, <italic toggle="yes">12</italic>). It is of
particular interest that
some of these adducts have been found not only in DNA of
animals treated with chemicals but also in DNA of animals
and humans not knowingly exposed to known precursors
(<italic toggle="yes">13</italic>), except perhaps certain chlorinated compounds
found
in disinfected drinking water (<italic toggle="yes">14</italic>).
<italic toggle="yes">N</italic><sup>2</sup>,3-ε-Gua,
1,<italic toggle="yes">N</italic><sup>6</sup>-ε-Ade,
3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyt, M<sub>1</sub>Gua, and
HO-propanoGua have all been found
at levels of 1 per 10<sup>6</sup>−10<sup>8</sup> bases in liver DNA
(<italic toggle="yes">13, 15</italic><italic toggle="yes">−</italic><italic toggle="yes">18</italic>).
The contribution of such bases to diseases such as
cancer
and to aging is a matter of interest (<italic toggle="yes">19, 20</italic>).
<fig id="bi972327rh00001" position="float" fig-type="scheme" orientation="portrait"><label>1</label><caption><p>Structures of (A) Five-Membered Ring Exocyclic Gua DNA Adducts Considered in This Work and (B) Other Adducts</p></caption><graphic xlink:href="bi972327rh00001.eps" position="float" orientation="portrait"></graphic></fig><fig id="bi972327rh00002" position="float" fig-type="scheme" orientation="portrait"><label>2</label><caption><p>Structures of Six-Membered Ring Exocyclic Gua DNA Adducts</p></caption><graphic xlink:href="bi972327rh00002.eps" position="float" orientation="portrait"></graphic></fig></p><p>Recently we have been interested in questions related to
mechanisms of misincorporation opposite the five-membered
exocyclic Gua adducts containing the ring structure shown
in Scheme <xref rid="bi972327rh00001"></xref>A [the so-called “linear” (as opposed to
“angular”) adducts (<italic toggle="yes">21</italic>)]. We previously quantified
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua in DNA treated with 2-chlorooxirane
(vinyl chloride epoxide) (<italic toggle="yes">22</italic>). Methods were developed
for
the synthesis of oligonucleotides containing each of these
two bases at a defined position, and in vitro
misincorporation
studies were done with several purified polymerases
(<italic toggle="yes">23</italic>).
The results collectively showed that both bases tend
to
strongly block replication at and beyond the site of
substitution and to favor the misincorporation of dATP and dGTP
across from both adducts. The selectivity of the
polymerases
varied. Analysis of steady-state kinetic results suggested
that
both <italic toggle="yes">Escherichia coli</italic> pol I (Kf<sup>-</sup>) and pol
II<sup>-</sup> misincorporated
>35% of the time, and frameshifts were also seen.
</p><p>In this work we examined miscoding opposite these two
adducts, 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethano Gua, in an
<italic toggle="yes">E. coli</italic>/M13MB19-based system that has also been used to study
the closely related six-membered ring homologues
M<sub>1</sub>Gua
and propanoGua (<italic toggle="yes">11, 12</italic>). The work was also extended
to
comparisons with the saturated model ethanoGua (Scheme
<xref rid="bi972327rh00003"></xref>). This adduct, which has not
been identified in DNA, was
used to provide in vivo and in vitro comparisons with the
other two five-membered ring exocyclic derivatives.
<fig id="bi972327rh00003" position="float" fig-type="scheme" orientation="portrait"><label>3</label><caption><p>Synthesis of EthanoGua</p></caption><graphic xlink:href="bi972327rh00003.eps" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e423"><title>Experimental Procedures</title><p><italic toggle="yes">Reagents</italic>. Reagents for oligonucleotide
synthesis were
purchased from PerSeptive Biosystems (Framingham, MA).
Polymerases (all exonuclease<sup>-</sup>) were purified by L.
L. Furge
(Department of Biochemistry) from recombinant expression
vectors described elsewhere (<italic toggle="yes">23</italic><italic toggle="yes">−</italic><italic toggle="yes">25</italic>),
and the extinction
coefficients cited were used to determine concentrations
of
diluted enzymes.
</p><p><italic toggle="yes">Instrumental Analysis</italic>. UV spectra were recorded
using
a modified Cary 14/OLIS instrument operating at ambient
temperature (On-Line Instrument Systems, Bogart, GA).
Positive ion electrospray mass spectra were obtained
using
direct loop injection into a Finnigan TSQ 7000 instrument
(Finnigan, Sunnyvale, CA). NMR spectra were recorded
in
the Vanderbilt facility using a Bruker AM-300 instrument
(Bruker, Billerica, MA). CGE was done with a Beckman
P/ACE 2000 or P/ACE 5000 instrument (Beckman, Fullerton,
CA) using a ssDNA 100 gel capillary. Samples were
applied
at −5 kV and run at −10 kV and 30 °C.
</p></sec><sec id="d7e444"><title></title><sec id="d7e446"><title>Synthesis of EthanoGua-Modified
Oligonucleotides</title><p><italic toggle="yes">EthanodGuo.</italic> The approach (Scheme <xref rid="bi972327rh00003"></xref>) was
patterned
after that used by Marinelli et al. (<italic toggle="yes">26</italic>) for
propanodGuo.
dGuo (270 mg, 1.0 mmol) and Na<sub>2</sub>CO<sub>3</sub>
(212 mg, 2.0 mmol,
finely ground with a pestle) were stirred in 4.0 mL of
(CH<sub>3</sub>)<sub>2</sub>SO under dry argon. 1,2-Dibromoethane (86 mL, 190 mg,
1.0 mmol) was added in a single portion, and the mixture
was stirred at room temperature overnight. The reaction
was
diluted with 2 vol of H<sub>2</sub>O, and the pH was adjusted to
∼5
with CH<sub>3</sub>CO<sub>2</sub>H. HPLC analysis was done
with a 10 × 250
mm Beckman Ultrasphere octadecylsilane column (5 μm;
Beckman, San Ramon, CA) and mixtures of two solvents,
(A) 50 mM NH<sub>4</sub>CH<sub>3</sub>CO<sub>2</sub>, pH
5.5, and (B) 90% CH<sub>3</sub>OH/10% H<sub>2</sub>O, v/v, with a flow rate of 4.0 mL
min<sup>-1</sup> and UV
detection at both 254 and 290 nm. The system was
programmed from a solvent mixture of 95% A/5% B (<italic toggle="yes">t</italic>
=
0) to 7% A/30% B (at 25 min) and then to 60% A/40% B
(at 30 min) (all v/v). Residual dGuo eluted at
<italic toggle="yes">t</italic><sub>R</sub> 12.5 min
(14% of the total <italic toggle="yes">A</italic><sub>254</sub> area), followed by two
major peaks
(<italic toggle="yes">t</italic><sub>R</sub> 17.2 and 30.7 min) and three minor ones.
The<italic toggle="yes"> t</italic><sub>R</sub> 17.2
min peak accounted for ∼50% of the total <italic toggle="yes">A</italic><sub>254</sub>
area and was
subsequently shown to be ethanodGuo. Further reaction
(to
40 h) did not appreciably change the HPLC profile.
The
gradient was modified slightly to improve
the preparative
separation of the peak of interest (85% A/15% B at <italic toggle="yes">t</italic> =
0,
78% A/22%B at 15 min, 60% A/40% B at 20 min, 85%
A/15% B at 25 min; elution of ethanodGuo at <italic toggle="yes">t</italic><sub>R</sub>
9.4 min).
CH<sub>3</sub>OH was removed in vacuo, and
NH<sub>4</sub>CH<sub>3</sub>CO<sub>2</sub> and
H<sub>2</sub>O
were removed by repeated lyophilization to yield 71 mg of
ethanodGuo, characterized by the following properties.
UV
(in HPLC solvent) λ<sub>max</sub>, nm: 253, 285. Positive
ion
electrospray mass spectrum, <italic toggle="yes">m</italic>/<italic toggle="yes">z</italic> 294.4 (rel
abundance 100,
MH<sup>+</sup>), 178.8 (24, M-116, loss of deoxyribose).
<sup>1</sup>H NMR
(300 MHz, (C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO) δ 2.22
(m, 1H, H-2‘ ‘), 2.48 (m, 1H,
H-2‘), 3.53 (m, 1H, H-5‘ ‘ or -5‘), 3.51 (m, 1H, H-5‘ ‘ or
-5‘ ‘),
3.54 (app t, 2H, −CH<sub>2</sub>− of ethano ring), 3.78 (m, 1H,
H-4‘),
4.04 (app t, 2H, −CH<sub>2</sub>− of ethano ring), 4.30 (m, 1H,
H-3‘),
4.95 (bs, 1H, 5‘-OH), 5.27 (bs, 1H, 3‘-OH), 6.09 (app t,
1H,
H-1‘), 7.81 (s, 1H, NH), 7.91 (s, 1H, H-2). <sup>13</sup>C NMR
(75
MHz, (C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO) δ 42.2
(C-3‘), 87.6 (C-1‘), 70.7 (C-10),
135.4 (C-2), 150.4 (C-3a), 155.4 and 155.5 (C-6 and C-9)
(the two ethano −CH<sub>2</sub>− peaks were not observed but
were
presumed to be obscured by the solvent, 38−40 ppm).
</p><p><italic toggle="yes">5</italic>‘<italic toggle="yes">-O-(4,4</italic>‘<italic toggle="yes">-Dimethoxytrityl)ethanodGuo.</italic>
The ethanodGuo
(57 mg, 0.19 mmol) from above was dried under vacuum in
a P<sub>2</sub>O<sub>5</sub> desiccator and then reacted with
4,4‘-dimethoxytrityl
chloride and <italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropylethylamine in
pyridine (6 mL)
under argon for 15 h at 4 °C using the general procedure
described elsewhere (<italic toggle="yes">27</italic>). A 2-fold excess of
4,4‘-dimethoxytrityl chloride and <italic toggle="yes">N,N</italic>-diisopropylethylamine was
then
added, and the reaction was allowed to proceed for another
4 h, after which the reaction was quenched with 26 mL of
CH<sub>3</sub>OH (with stirring for 10 min) and concentrated
to
dryness in vacuo. The residue was dissolved in
CH<sub>2</sub>Cl<sub>2</sub> and
washed with 10% aq K<sub>2</sub>CO<sub>3</sub> (w/v), and the
product was
purified by flash chromatography on silica gel, eluting
with
a mixture of
CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH/(C<sub>2</sub>H<sub>5</sub>)<sub>3</sub>N
(95:5:0.3, v/v/v). The
fractions containing the product, as judged by TLC, were
combined and concentrated in vacuo to yield 51 mg of 5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)ethanodGuo (48% yield).
<sup>1</sup>H NMR
(C<sup>2</sup>HCl<sub>3</sub>) δ 2.44 (m, 1H, H-2‘ ‘), 2.69 (m,
1H, H-2‘), 3.32
(m, 1H, H-5‘ or -5‘ ‘), 3.67 (m, 1H, H-5‘ or -5‘ ‘), 3.73
(app
t, 2H, −CH<sub>2</sub>− of ethano ring), 4.12 (m, 1H, H-4‘), 4.23
(t,
2H, −CH<sub>2</sub>- of ethano ring), 4.60 (m, 1H, H-3‘), 5.15 (s,
1H,
3‘-OH), 6.19 (s, 1H, 1‘-OH), 6.80 and 6.82 (s, 3H,
−OCH<sub>3</sub>
groups), 7.22−7.42 (aromatic region), 7.59 (s, 1H H-2).
</p><p><italic toggle="yes">3</italic>‘<italic toggle="yes">-O-[(N,N-Diisopropylamino)</italic><italic toggle="yes">-</italic><italic toggle="yes">(2-cyanoethyl)phosphoinyl]-5</italic>‘<italic toggle="yes">-O-4,4</italic>‘<italic toggle="yes">-</italic><italic toggle="yes">dimethoxytrityl)ethanodGuo.</italic>
The 5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)ethanodGuo from above (16.2 mg, 26
μmol)
was dried by using repeated evaporation from anhydrous
pyridine and reacted with 1<italic toggle="yes">H</italic>-tetrazole (6.9 mg, 31
μmol,
1.2 equiv) and 2-cyano-
<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>‘,<italic toggle="yes">N</italic>‘-tetraisopropylphosporamidite (12 μL, 37 mg, 38 μmol, 1.5 equiv) in 2 mL of
CH<sub>2</sub>Cl<sub>2</sub> for 2 h (<italic toggle="yes">27</italic>), and the
progress of the reaction was
monitored by TLC. The product was purified by
extraction
(with 10% aq NaHCO<sub>3</sub>, w/v, from
CH<sub>2</sub>Cl<sub>2</sub>) and flash
chromatography
(CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH/(C<sub>2</sub>H<sub>5</sub>)<sub>3</sub>N,
95:5:0.5, v/v/v) to yield 29 mg of the phosphoramidite. <sup>31</sup>P
NMR
(C<sup>2</sup>H<sub>3</sub>CN) δ 27.83.
</p><p>The product was dissolved in CH<sub>3</sub>CN and used in
the
synthesis of the 8-mer d(5‘-GGTG*TCCG-3‘) and the 19-mer d(5‘-CAGTGGGTG*TCCGAATTGA-3‘) for these studies (G* = ethanoGua). The coupling efficiency was
∼50%
at the site of addition in both cases. The
oligonucleotides
(with 4-<italic toggle="yes">tert</italic>-butylphenoxyacetyl protecting groups)
were
treated with 0.10 N NaOH at room temperature overnight
to deprotect.
</p><p><italic toggle="yes">Oligonucleotide Purification.</italic> The different
oligonucleotides were purified using a 10 × 250 mm octadecylsilane
reversed-phase HPLC column (YMC-Pack ODS-AQ, 5 μm,
YMC, Wilmington, NC), first with a linear gradient of
0−30% CH<sub>3</sub>CN (v/v) in 50 mM
NH<sub>4</sub>CH<sub>3</sub>CO<sub>2</sub> buffer,
pH
5.0, over 50 min. Solvent was removed in vacuo and by
lyophilization, and the oligonucleotides were further
purified
using a second linear gradient of 20−26% CH<sub>3</sub>CN
(v/v) in
the same buffer over 50 min. The oligonucleotides
were
then purified on 20% polyacrylamide (w/v) gels buffered
with sodium MOPS (pH 7.0). Purity of all
oligonucleotides
was checked by CGE, and purity of >99% was achieved
before use (Figure <xref rid="bi972327rf00001"></xref>, vide infra).
</p><p><italic toggle="yes">Characterization of Modified Oligonucleotides</italic>. The
10-mer “primer” oligonucleotide d(5‘-TCAATTCGGA-3‘) was
prepared by automated synthesis and purified as described
previously (<italic toggle="yes">23</italic>). The 8-mer d(5‘-GGTGTCCG-3‘)
was
prepared in the same manner: ES MS <italic toggle="yes">m</italic>/<italic toggle="yes">z</italic> 2441.0
(MH<sup>-</sup>),
theor 2441.6. The 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua monomers
were synthesized as described and used to prepare the same
8-mer with the most central Gua substituted.
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
8-mer, ES MS <italic toggle="yes">m</italic>/<italic toggle="yes">z</italic> 2465.0 (MH<sup>-</sup>),
theor 2465.0. HO-ethanoGua 8-mer, ES MS <italic toggle="yes">m</italic>/<italic toggle="yes">z</italic> 2483.2
(MH<sup>-</sup>), theor 2483.0.
The 19-mer “template” oligomers,
d(5‘-CAGTGGGTG*TCCGAATTGA-3‘) (G* = modified G), were prepared as
described previously (<italic toggle="yes">23</italic>).
</p><p>The 8-mer and 19-mer containing ethanoGua were characterized after digestion with 8 μg of nuclease P<sub>1</sub>
(Sigma
Chemical Co., St. Louis, MO) at 37 °C for 3 h followed
by
digestion with 9 μg of snake venom phosphodiesterase
(Sigma) and 6 μg of alkaline phosphatase (Sigma) at 37
°C
for another 3 h. Digested products were then analyzed
by
HPLC using a 10 × 250 mm YMC octadecylsilane column
(vide supra) and a gradient formed with 50 mM
NH<sub>4</sub>CH<sub>3</sub>CO<sub>2</sub>, pH 4.5, (A) and CH<sub>3</sub>CN (B): 0 min
(100% A, 0% B),
25 min (90% A, 10% B), 35 min (80% A, 20% B), 45 min
(50% A, 50% B), 55 min (50% A, 50% B), and 60 min
(100% A, 0% B) (flow 1.0 mL min<sup>-1</sup>).
Under these
conditions ethanodGuo was eluted at <italic toggle="yes">t</italic><sub>R</sub> 38 min
(Figure <xref rid="bi972327rf00002"></xref>).
</p><p>For all oligonucleotides, extinction coefficients were
estimated using the method of Borer (<italic toggle="yes">28</italic>).
</p></sec></sec><sec id="d7e877"><title></title><sec id="d7e879"><title>General Methods for Site-Specific Mutagenesis</title><p><italic toggle="yes">Bacterial Strains</italic>. Three<italic toggle="yes"> E. coli</italic> strains
were used in this
study: LM102 (AB1157 F‘ [<italic toggle="yes">traD36 proAB
lacIQlacZ</italic>Δ<italic toggle="yes">M</italic><italic toggle="yes">15</italic>]),
LM103 (AB1157 uvrA6 F‘ [<italic toggle="yes">traD36 </italic><italic toggle="yes">proAB
lacIQlacZ</italic>Δ<italic toggle="yes">M</italic><italic toggle="yes">15</italic>]),
and JM105 (<italic toggle="yes">supE thi rspL endA sbcB15 hsd R4
</italic>Δ<italic toggle="yes">(lac-</italic><italic toggle="yes">proAB)
F</italic>‘<italic toggle="yes"> [traD36 proAB
lacIQZ</italic>Δ<italic toggle="yes">M</italic><italic toggle="yes">15]))</italic>.
</p><p><italic toggle="yes">M13MB102 Preparation</italic>. The general approach
described
by Benamira and Marnett (<italic toggle="yes">29, 30</italic>) was used.
Briefly,
bacteriophage M13MB102 corresponds to a M13mp19
genome in which 27 bp of the <italic toggle="yes">his</italic>D3052 sequence
of
<italic toggle="yes">Salmonella typhimurium</italic> has been incorporated at
position
6256 (<italic toggle="yes">30</italic>). Single-stranded M13MB102 was grown in
the
presence of JM105, and the Qiagen (Chatsworth, CA) Maxi-prep procedure was followed to extract double-stranded
DNA, while single-stranded DNA was recovered by precipitation with 2.5% poly(ethylene glycol) 8000
(w/v).
</p><p><italic toggle="yes">DNA Construction, Transfection, and Mutagenesis</italic>.
Gapped
duplex DNA was obtained by dialysis of DNA linearized
by <italic toggle="yes">Bss</italic>HII and <italic toggle="yes">Ksp</italic>I
in the presence of a 12-fold excess of
single-stranded DNA, from 95% (v/v) to 0% formamide
(<italic toggle="yes">30</italic>).
Phosphorylated 8-mer oligonucleotides (containing or
not
containing the adduct) were ligated into the gapped duplex
for 4 h at 16 °C with 400 units of DNA ligase
(Boehringer-Mannheim, Indianapolis, IN) in 50 mM sodium MOPS buffer
(pH 7.0). Ligated products were purified by
centrifugation
through Ultrafree Probind filters (0.45 μm, Millipore,
Bedford, MA), recovered from a 0.8% (w/v) low-melting-point
agarose gel with 5 units of β-agarase (New England
Biolabs,
Beverly, MA), and concentrated with a Microcon 3 device
(Amicon, Beverly, MA) following the manufacturer's instructions. <italic toggle="yes">E. coli</italic> cells were grown to mid-log
phase,
isolated, resuspended, and irradiated with a hand-held UV
light (254 nm) to induce the SOS system, as previously
described (<italic toggle="yes">33</italic>). The dose of UV was determined in
separate
experiments (varying time from 0 to 3 min) to reduce
bactrial
survival to 1−10%. Transfection of DNA into <italic toggle="yes">E. coli</italic>
was
performed by electroporation using a Gibco BRL
Cell-porator<italic toggle="yes">
E. coli</italic> system (Gaithersburg, MD). Bacteria were
plated
on LB plates in the presence of isopropyl
β-<sc>d</sc>-thiogalactoside
(<italic toggle="yes">31</italic>) and grown overnight. The phage population
was
recovered by shaking the plates in 10 mM Tris buffer
containing 1.0 mM EDTA (pH 7) for 3 h. <italic toggle="yes">E. coli</italic>
JM105
was then infected with the phage DNA population and plated
in the presence of 5-bromo-4-chloro-3-indolyl
β-<sc>d</sc>-galactopyranoside (Sigma Chemical Co., St. Louis, MO) and
isopropyl β-<sc>d</sc>-thiogalactoside to obtain 300−400 blue
plaques
per plate.
</p><p><italic toggle="yes">Hybridization</italic>. Single-stranded DNA (+ strand) was
lifted
onto nitrocellulose membranes (Schleicher and Schull,
Keene, NH) after chilling for 2 h and then baked under
vacuum at 80 °C for 2 h. Hybridizations were performed
at
32 °C with the following hybridization media: 2×
SSPE,
20% formamide (v/v), 10% SDS (w/v), 2.5 mg/mL calf
thymus DNA, and 2.5% Denhart's solution (<italic toggle="yes">32</italic>).
Labeled
13-mer oligonucleotides bearing the four different
nucleotides
opposite position 6256 were used as specific probes and
designated as follows: probe A (for G→A mutations),
d(5‘-GCCGCGGT<italic toggle="yes">A</italic>TCCG-3‘); probe G (wild type), d(5‘-GCCGCGGT<italic toggle="yes">G</italic>TCCG-3‘); probe C (G→C), d(5‘-GCCGCGGT<italic toggle="yes">C</italic>TCCG-3‘); and probe T (G→T),
d(5‘-GCCGCGGT<italic toggle="yes">T</italic>TCCG-3‘). Finally, washes were performed in 2×
SSC
twice for 20 min at the hybridization temperature, and the
membranes were exposed to X-ray film for 6−24 h at −80
°C.
</p><p><italic toggle="yes">T Construct</italic>. To study the strand utilization and
estimate
the blockage of replication that occurred in the presence
of
the adducts, another vector, termed the “T construct” in
this
study, was used. This construct differed by the presence
of
a T in the plus strand at position 6256, corresponding to
the
adduct site (<italic toggle="yes">12, 33</italic>). The same procedure was used
with
this construct as with M13MB102, except that only hybridizations with the A probe were performed.
</p><p><italic toggle="yes">In Vitro</italic> <italic toggle="yes">Experiments</italic>. The general
experimental procedures used were as described previously for the related ε
adducts (<italic toggle="yes">23</italic>). Briefly, a 10-mer primer (2 μM) was
5‘-labeled
with T4 kinase (Boehringer-Mannheim), annealed at 90 °C
for 10 min, and slowly cooled to room temperature with a
19-mer template containing Gua or ethanoGua at position
11. Either a mixture of of the four dNTPs (100 μM) or
a
single dNTP (100 μM) was incubated in 50 mM sodium
MOPS buffer (pH 7.0) containing 2 μg/mL bovine serum
albumin, 4 mM dithiothreitol, and 8 mM MgCl<sub>2</sub> in
the
presence of one of the polymerases.
</p><p>The concentration of a single dNTP was varied to
determine <italic toggle="yes">k</italic><sub>cat</sub> and <italic toggle="yes">K</italic><sub>m</sub>,
which were estimated from measurements of the percent extension of the primer with a
nonlinear
fitting program (k·cat, BioMetallics, Princeton, NJ),
using
an approach patterned after that of Boosalis et al.
(<italic toggle="yes">34</italic>).
Concentrations of polymerases and reaction times were
varied to keep the extent of primer extension less than
∼20%.
The products were analyzed on 20% (w/v) denaturing
polyacrylamide−urea gels and exposed using a Molecular
Dynamics model 400E Phosphorimager (Molecular Dynamics Inc., Sunnyvale, CA). Representative details of
concentrations and time are presented in the appropriate tables
and
figures.
</p><p>Sequence analyses were performed by following the
procedure described by Maxam and Gilbert (<italic toggle="yes">35, 36</italic>)
except
for the T-specific reaction, where the modification
described
by Friedman and Brown (<italic toggle="yes">37</italic>) was used.
</p></sec></sec><sec id="d7e1045"><title>Results</title><p><italic toggle="yes">Mutagenicity of
1,N</italic><italic toggle="yes"><sup>2</sup></italic><sup></sup><italic toggle="yes">-</italic>ε<italic toggle="yes">-Gua and
HO-EthanoGua in E. coli.</italic>
Oligonucleotides containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
and HO-ethanoGua
were synthesized and characterized as described previously
(<italic toggle="yes">23</italic>), ligated into an M13MB102 gap duplex
genome,
transfected into LM102 (a wild-type <italic toggle="yes">E. coli</italic> strain),
and
compared to a construct containing Gua at the same
position
(6256). When 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua was substituted
for G, the
frequency of mutation to A was 0.34%, the frequency of
mutation to T was 0.25%, and mutation to C was undetectable (Table <xref rid="bi972327rt00001"></xref>). Lower mutation
frequencies were observed
when HO-ethanoGua was incorporated into the same G site
of M13MB102 phage (0.14 ± 0.09% A, 0.09 ± 0.034% T,
and 0.12 ± 0.04% C). These changes were all highly
dependent upon induction of the SOS response by UV
irradiation. Without induction, no mutants were
observed
after hybridization for any of the three
misincorporation
products opposite the adduct.
<table-wrap id="bi972327rt00001" position="float" orientation="portrait"><label>1</label><caption><p>Percentages of Base-Pair Substitutions opposite Gua and Gua Derivatives in Pre-Irradiated<italic toggle="yes">E.
</italic><italic toggle="yes">c</italic><italic toggle="yes">oli</italic><italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="5"><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:tbody><oasis:row><oasis:entry colname="1">base</oasis:entry><oasis:entry colname="2">uvrA
phenotype</oasis:entry><oasis:entry colname="3">G→A,
A probe (%)</oasis:entry><oasis:entry colname="4">G→T,
T probe (%)</oasis:entry><oasis:entry colname="5">G→C,
C probe (%)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3"><0.09
</oasis:entry><oasis:entry colname="4"><0.07
</oasis:entry><oasis:entry colname="5"><0.10
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3">0.34 ± 0.16
</oasis:entry><oasis:entry colname="4">0.25 ± 0.12
</oasis:entry><oasis:entry colname="5"><0.08
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3"><0.10
</oasis:entry><oasis:entry colname="4"><0.05
</oasis:entry><oasis:entry colname="5"><0.11
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3">2.05 ± 0.20
</oasis:entry><oasis:entry colname="4">0.74 ± 0.25
</oasis:entry><oasis:entry colname="5">0.09 ± 0.02
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3"><0.09
</oasis:entry><oasis:entry colname="4"><0.09
</oasis:entry><oasis:entry colname="5"><0.11
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3">0.14 ± 0.09
</oasis:entry><oasis:entry colname="4">0.09 ± 0.03
</oasis:entry><oasis:entry colname="5">0.12 ± 0.04
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3"><0.07
</oasis:entry><oasis:entry colname="4"><0.07
</oasis:entry><oasis:entry colname="5"><0.07
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3">0.11 ± 0.04
</oasis:entry><oasis:entry colname="4">0.19 ± 0.09
</oasis:entry><oasis:entry colname="5">0.07 ± 0.04
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3"><0.08
</oasis:entry><oasis:entry colname="4"><0.18
</oasis:entry><oasis:entry colname="5"><0.09
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">ethanoGua
</oasis:entry><oasis:entry colname="2">+
</oasis:entry><oasis:entry colname="3">0.17 ± 0.14
</oasis:entry><oasis:entry colname="4">0.11 ± 0.05
</oasis:entry><oasis:entry colname="5"><0.08
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3"><0.07
</oasis:entry><oasis:entry colname="4"><0.07
</oasis:entry><oasis:entry colname="5"><0.06
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">ethanoGua
</oasis:entry><oasis:entry colname="2">−
</oasis:entry><oasis:entry colname="3">0.71 ± 0.18
</oasis:entry><oasis:entry colname="4">0.71 ± 0.23
</oasis:entry><oasis:entry colname="5"><0.19</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Results are expressed as percent mutation
frequencies ± SD and
were obtained by screening ∼1000 plaques per experiment for
each
probe. Values are the average of 2−9 independent experiments.
An
experiment with Gua was done at the same time as each
experiment
with a Gua derivative. The “<” designations indicate that no
mutations
were observed at the indicated limit of detection. Limits of
detection
differ because of differences in the number of plaques
among
experiments. Values for G probes approach 100% but are not
shown.
Values for nonirradiated cells were all in the range of <0.06 to
<0.18
in all cases but are not shown.</p></table-wrap-foot></table-wrap></p><p>The uvrA protein plays an essential role in the nucleotide
excision repair system in wild-type <italic toggle="yes">E. coli</italic>, and
comparisons
were made with a deficient strain, LM103. No mutants
were
detectable without induction (of the SOS system) (Table
<xref rid="bi972327rt00001"></xref>).
With induction, 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua increased the
G→A mutation
frequency to 2.05%, the G→T frequency was 0.74%, and
the G→C frequency was still very low (0.09 ± 0.02%).
Interestingly, no significant increase of the overall
mutation
frequency due to HO-ethanoGua was detectable in the uvrA-deficient strain.
</p><p><italic toggle="yes">Synthesis of EthanoGua, the Saturated
</italic><italic toggle="yes">Homologue</italic><italic toggle="yes"> of
1,N</italic><italic toggle="yes"><sup>2</sup></italic><sup></sup><italic toggle="yes">-</italic>ε<italic toggle="yes">-Gua.</italic> Different mutation results were observed
with 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua, which formally differ only by a
molecule of H<sub>2</sub>O (Table <xref rid="bi972327rt00001"></xref>). A third exocyclic Gua
adduct,
ethanoGua, the saturated homologue of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (Scheme
<xref rid="bi972327rh00001"></xref>), was examined. dGuo was modified to form
ethanodGuo
(Scheme <xref rid="bi972327rh00003"></xref>), which was incorporated into oligonucleotides
after protection of the hydroxyls with dimethoxytrityl and
phosphoramidite groups. After deprotection, HPLC, and
electrophoretic purification, purity was evaluated using
two
different techniques: (i) <sup>32</sup>P-labeling of
oligonucleotides with
T4 polynucleotide kinase and analysis on 20% acrylamide
gels and (ii) CGE. Three preparations of the 8-mer oligonucleotide containing ethanoGua are shown (Figure
<xref rid="bi972327rf00001"></xref>).
Electrophoretic analysis showed a single band in all
three
cases. In contrast, CGE analysis demonstrated that
preparation a contained an impurity and preparation b was a
mixture
of two major peaks. Preparation c was of high purity.
The
8-mer and 19-mer oligonucleotides containing ethanoGua
were digested with nucleases and phosphatases, and the
products were analyzed by HPLC (Figure
<xref rid="bi972327rf00002"></xref>). The impurities
in preparations a and b were not identified. Preparation
c
was ligated into the vector for mutational analysis.
These
and other observations with oligonucleotides indicate that
caution needs to be taken regarding purity of
oligonucleotides
used in such studies, particularly in
vivo work, and underline
the necessity of using high-resolution, independent
techniques
such as CGE to verify the purity of oligonucleotides
following separations.
<fig id="bi972327rf00001" position="float" orientation="portrait"><label>1</label><caption><p>CGE profiles of oligonucleotides containing ethanoGua. In panel A, three preparations of an 8-mer containing ethanoGua were end-labeled with<sup>32</sup>P and subjected to electrophoresis in a
polyacrylamide-urea gel (20%, w/v), and the results were visualized
by
phosphorimaging. The three preparations are labeled a, b, and c and
were analyzed by CGE in panels B, C, and D, respectively.
The
conditions were different in the three CGE runs, which were done at
different times, so the migration times are not directly
comparable.
Only preparation c was used for mutagenesis studies.
</p></caption><graphic xlink:href="bi972327rf00001.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi972327rf00002" position="float" orientation="portrait"><label>2</label><caption><p>Characterization of the 8-mer d(5‘-GGTGTCCG-3‘) containing ethanoGua. The 8-mer was digested enzymatically as described in Experimental Procedures. (A) HPLC with no materials injected. (B) HPLC of a digest of the 8-mer. Standard deoxyribonucleosides were used to identify the peaks in a separate HPLC separation (not shown).</p></caption><graphic xlink:href="bi972327rf00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Misincorporation of dNTPs opposite EthanoGua in the
Presence of Purified </italic><italic toggle="yes">Polymerases</italic>. Extension of a
10-mer
primer annealed with a 19-mer template containing
ethanoGua was compared with an unmodified template (Scheme
<xref rid="bi972327rh00004"></xref>) using each of four polymerases, Kf<sup>-</sup>, pol
II<sup>-</sup>, T7<sup>-</sup>, and
HIV RT, as in a previous study (<italic toggle="yes">23</italic>). Full-length
extension
(in the presence of the four dNTPs) was highly retarded
with
all four polymerases (Figure <xref rid="bi972327rf00003"></xref>).
Blockage of extension by
ethanoGua was more extensive than in the case of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua. Some 11-mer product was observed with Kf<sup>-</sup> and,
to
a lesser extent, with pol II<sup>-</sup>, corresponding to blockage
after
incorporation of one base opposite the adduct (Scheme
<xref rid="bi972327rh00004"></xref>).<xref rid="bi972327rX00002" ref-type="bibr"></xref><fig id="bi972327rf00003" position="float" orientation="portrait"><label>3</label><caption><p>Extension of 10-mer primers by polymerases in the presence of all four dNTPs and various 19-mers containing Gua, 1,<italic toggle="yes">N</italic><sup>2</sup>ε-Gua,
or ethanoGua at position 11. Results are shown for the unmodified
19-mer and the 19-mer templates containing
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and ethanoGua,
with varying concentrations of (A) Kf<sup>-</sup>, (B) pol
II<sup>-</sup>, (C) T7<sup>-</sup>, and (D) HIV RT. The reaction
time was 30 min in all cases, and 100 μM each
of dATP, dCTP, dGTP, and dTTP was present.</p></caption><graphic xlink:href="bi972327rf00003.eps" position="float" orientation="portrait"></graphic></fig><fig id="bi972327rh00004" position="float" fig-type="scheme" orientation="portrait"><label>4</label><caption><p>Oligonucleotides Used in EthanoGua Misincorporation Assays</p></caption><graphic xlink:href="bi972327rh00004.eps" position="float" orientation="portrait"></graphic></fig></p><p>Full-length extension products obtained by primer extension in the presence of all four dNTPs were sequenced
(<italic toggle="yes">35,
36</italic>). Analysis clearly showed that the 19-mer
product
obtained by extension in the presence of Kf<sup>-</sup>
corresponded
to an incorporation of G opposite the adduct, while A was
incorporated at this site in the presence of pol II<sup>-</sup> or
T7<sup>-</sup>
(data not shown), as reported earlier for
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua (<italic toggle="yes">23</italic>) and also seen with HIV RT and
<italic toggle="yes">S</italic>-glutathionylethyl-substituted Gua
derivatives.<xref rid="bi972327rX00003" ref-type="bibr"></xref></p><p>Primer extension assays were done in the presence of
individual dNTPs (Figure <xref rid="bi972327rf00004"></xref>), and G was
mainly incorporated
in the presence of Kf<sup>-</sup>; C and T were also incorporated.
The
incorporation of more than one base with dCTP, yielding a
13-mer product, can be explained by a slippage to the
three
following G's present (two nucleotides after the adduct)
(Scheme <xref rid="bi972327rh00004"></xref>). Similar experiments were
performed in the
presence of pol II<sup>-</sup> and T7<sup>-</sup> (Table
<xref rid="bi972327rt00002"></xref>); A was mainly
incorporated, as well as some C in the presence of pol
II<sup>-</sup>
and some G in the case of T7<sup>-</sup>.
<fig id="bi972327rf00004" position="float" orientation="portrait"><label>4</label><caption><p>Extension of 10-mer primer with Kf<sup>-</sup> in the
presence
of single dNTPs.</p></caption><graphic xlink:href="bi972327rf00004.eps" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi972327rt00002" position="float" orientation="portrait"><label>2</label><caption><p>Products of Polymerase Extension Assays of 10-mer Primer in the Presence of All Four dNTPs (Extension Assays) or a Single dNTP (1-Base Incorporation) in the Presence of 19-mers</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><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:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="4">products</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">polymerase</oasis:entry><oasis:entry colname="3">base = Gua</oasis:entry><oasis:entry colname="4">base = ethanoGua
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">extension assays
</oasis:entry><oasis:entry colname="2">Kf<sup>-</sup></oasis:entry><oasis:entry colname="3">19 + 20 mer
</oasis:entry><oasis:entry colname="4">11 + 17 + 18 + 19 + 20 mer
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">pol II<sup>-</sup></oasis:entry><oasis:entry colname="3">18 + 19 mer
</oasis:entry><oasis:entry colname="4">18 + 19 mer
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">T7<sup>-</sup></oasis:entry><oasis:entry colname="3">19 + 20 mer
</oasis:entry><oasis:entry colname="4">19 + 20 mer
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">HIV RT
</oasis:entry><oasis:entry colname="3">18 + 19 mer
</oasis:entry><oasis:entry colname="4">9 + 8 + 7 mer
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1-base
</oasis:entry><oasis:entry colname="2">Kf<sup>-</sup></oasis:entry><oasis:entry colname="3">C > A, G, T
</oasis:entry><oasis:entry colname="4">G > T, C
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">incorporation</oasis:entry><oasis:entry colname="2">pol II<sup>-</sup></oasis:entry><oasis:entry colname="3">C > A, G, T
</oasis:entry><oasis:entry colname="4">A, C
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">T7<sup>-</sup></oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">A, G
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">HIV RT
</oasis:entry><oasis:entry colname="3">−
</oasis:entry><oasis:entry colname="4">−</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table></table-wrap></p><p>Steady-state kinetic assays of single dNTP incorporation
were used to characterize incorporation parameters more
quantitatively (Table <xref rid="bi972327rt00003"></xref>; Figure
<xref rid="bi972327rf00005"></xref>). In the presence of
Kf<sup>-</sup>,
a significant decrease of <italic toggle="yes">K</italic><sub>m</sub> was observed when
dGTP or
dTTP was incorporated, compared to the normal base dCTP.
In contrast, <italic toggle="yes">k</italic><sub>cat</sub> values with
Kf<sup>-</sup> were similar for dCTP and
dTTP incorporation and increased slightly for dGTP.
With
pol II<sup>-</sup>, the <italic toggle="yes">k</italic><sub>cat</sub> value for dATP
incorporation opposite the
adduct was increased compared to dCTP, and the
<italic toggle="yes">K</italic><sub>m</sub> values
were similar for the two nucleotides. When
comparisons
are made between rates of incorporation of dNTPs opposite
ethanoGua with pol II<sup>-</sup>, the <italic toggle="yes">k</italic><sub>cat</sub>
values are similar to that
seen for misincorporation of dATP opposite Gua. With
Kf<sup>-</sup>,
the <italic toggle="yes">k</italic><sub>cat</sub> values were all lower for all
incorporations opposite
ethanoGua than for any dNTPs opposite Gua.
<italic toggle="yes">K</italic><sub>m</sub> values
varied considerably. The misincorporation ratios
[defined
as
(<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 (<italic toggle="yes">37</italic>)] were
all very high compared to the values obtained for
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua under the same conditions [0.15−1.2 (<italic toggle="yes">23</italic>)] and indicate a strong preference against
incorporation of dCTP.
<fig id="bi972327rf00005" position="float" orientation="portrait"><label>5</label><caption><p>Incorporation of dNTPs opposite ethanoGua by Kf<sup>-</sup>
as
a function of dNTP concentration. Incubations were done with
80
nM primer−template concentration for 30 min with the
indicated
concentration of each dNTP. The concentration of Kf<sup>-</sup> was
25 nM
in the experiments with dGTP and dTTP and 75 nM in the
experiments with dCTP. The percent primer extension,
determined
by phosphorimager analysis, is shown as the mean ± SD of
three
determinations at each dNTP concentration, without correction
for
the enzyme concentration. Calculated kinetic parameters:
dCTP,
<italic toggle="yes">k</italic><sub>cat</sub> 9.9(±0.9) ×
10<sup>-4</sup> min<sup>-1</sup>,
<italic toggle="yes">K</italic><sub>m</sub> 950(±190) μM; dGTP,
<italic toggle="yes">k</italic><sub>cat</sub> 25(±2)
× 10<sup>-4</sup> min<sup>-1</sup>,
<italic toggle="yes">K</italic><sub>m</sub> 45(±9) μM; dTTP,
<italic toggle="yes">k</italic><sub>cat</sub> 4.5(±0.2) ×
10<sup>-4</sup>
min<sup>-1</sup>,
<italic toggle="yes">K</italic><sub>m</sub> 38(±7) μM (Table <xref rid="bi972327rt00003"></xref>).</p></caption><graphic xlink:href="bi972327rf00005.eps" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi972327rt00003" position="float" orientation="portrait"><label>3</label><caption><p>Steady-State Kinetic Parameters for dNTP Incorporation opposite EthanoGua</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><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:tbody><oasis:row><oasis:entry colname="1">polymerase</oasis:entry><oasis:entry colname="2">template base</oasis:entry><oasis:entry colname="3">substrate</oasis:entry><oasis:entry colname="4"><italic toggle="yes">k</italic><sub>cat</sub> (min<sup>-1</sup>)</oasis:entry><oasis:entry colname="5"><italic toggle="yes">K</italic><sub>m</sub> (μM)</oasis:entry><oasis:entry colname="6">misincorporation ratio<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Kf<sup>-</sup></oasis:entry><oasis:entry colname="2">ethanoGua
</oasis:entry><oasis:entry colname="3">dCTP
</oasis:entry><oasis:entry colname="4">9.9(±0.9) × 10<sup>-4</sup></oasis:entry><oasis:entry colname="5">950 ± 190
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ethanoGua
</oasis:entry><oasis:entry colname="3">dGTP
</oasis:entry><oasis:entry colname="4">24.7(±1.7) × 10<sup>-4</sup></oasis:entry><oasis:entry colname="5">45 ± 9
</oasis:entry><oasis:entry colname="6">53
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ethanoGua
</oasis:entry><oasis:entry colname="3">dTTP
</oasis:entry><oasis:entry colname="4">4.5(±0.2) × 10<sup>-4</sup></oasis:entry><oasis:entry colname="5">38 ± 7
</oasis:entry><oasis:entry colname="6">11
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">Gua
</oasis:entry><oasis:entry colname="3">dCTP
</oasis:entry><oasis:entry colname="4">16 ± 5
</oasis:entry><oasis:entry colname="5">0.044 ± 0.024
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">Gua
</oasis:entry><oasis:entry colname="3">dGTP
</oasis:entry><oasis:entry colname="4">3.8(±0.4) × 10<sup>-1</sup></oasis:entry><oasis:entry colname="5">39 ± 7
</oasis:entry><oasis:entry colname="6">2.7 × 10<sup>-5</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">Gua
</oasis:entry><oasis:entry colname="3">dTTP
</oasis:entry><oasis:entry colname="4">1.8(±0.3) × 10<sup>-1</sup></oasis:entry><oasis:entry colname="5">28 ± 11
</oasis:entry><oasis:entry colname="6">1.8 × 10<sup>-5</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol II<sup>-</sup></oasis:entry><oasis:entry colname="2">ethanoGua
</oasis:entry><oasis:entry colname="3">dCTP
</oasis:entry><oasis:entry colname="4">1.9(±0.2) × 10<sup>-4</sup></oasis:entry><oasis:entry colname="5">26 ± 10
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">ethanoGua
</oasis:entry><oasis:entry colname="3">dATP
</oasis:entry><oasis:entry colname="4">5.1(±0.5) × 10<sup>-4</sup></oasis:entry><oasis:entry colname="5">11 ± 4
</oasis:entry><oasis:entry colname="6">6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">Gua
</oasis:entry><oasis:entry colname="3">dCTP
</oasis:entry><oasis:entry colname="4">15 ± 2
</oasis:entry><oasis:entry colname="5">3 ± 1
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">Gua
</oasis:entry><oasis:entry colname="3">dATP
</oasis:entry><oasis:entry colname="4">3.5(±1.2) × 10<sup>-3</sup></oasis:entry><oasis:entry colname="5">287 ± 111
</oasis:entry><oasis:entry colname="6">2.5 × 10<sup>-6</sup></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Misincorporation 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 (<italic toggle="yes">34</italic>) (also termed the misincorporation
frequency in this and other
references). Results indicate SE within experiments as calculated by
the analysis program. The values for dCTP incorporation include
incorporation
of 2- and 3-base addition products that occur due to slipped
intermediates (<italic toggle="yes">23</italic>), and values for dATP include the 2-base
extension product that
results because of the rapid addition of an additional dATP opposite
the T 3‘ of the ethanoGua adduct. See Figure <xref rid="bi972327rf00005"></xref> for plots of results
obtained
with Kf<sup>-</sup> and ethanoGua. In experiments done with pol
II<sup>-</sup> and ethanoGua, the concentration of pol
II<sup>-</sup> was 5 nM, the concentration of primer−template complex was 100 nM, and the dNTP concentrations were 1, 5,
10.25, 50, 75, and 100 μM; the reaction time was 30 min. In
experiments
with Kf<sup>-</sup> and Gua-containing template (100 nM), the
concentration of Kf<sup>-</sup> was 1 nM in the case of dCTP
(reaction time 5 min); with dGTP and
dTTP the concentration of Kf<sup>-</sup> was 10 nM, and the reaction
time was 30 min. With pol II<sup>-</sup> and the Gua-containing
template (100 nM), the
concentration of pol II<sup>-</sup> was 0.1 nM with dCTP (reaction
time 5 min) and 100 nM with dATP (reaction time 30
min).</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">EthanoGua-Induced Mutations in E. coli.</italic> Both
LM102,
the wild-type strain for DNA repair, and LM103, the uvrA-deficient strain, were transformed with phage DNA containing ethanoGua or Gua at position 6256 of the minus strand.
No mutations were detectable without SOS induction
(by
UV light) in either the LM102 or the LM103 <italic toggle="yes">E. coli</italic>
strain
(Table <xref rid="bi972327rt00001"></xref>). After induction, G→A (0.17%) and G→T
mutations (0.11%) were detected with the ethanoGua
derivative in <italic toggle="yes">E. coli</italic> LM102. Significant increases of mutation
to
T and A at the adduct site were observed in the nucleotide
excision repair deficient background (Table <xref rid="bi972327rt00001"></xref>). The
mutation
frequencies reached equivalent values for G→A and G→T
mutation frequencies (0.71% each). In contrast, the
G→C
mutation frequency (corresponding to G incorporation opposite the adduct) remained below the limit of detection.
</p><p><italic toggle="yes">Effect of </italic><italic toggle="yes">Five</italic><italic toggle="yes">-Membered Exocyclic Ring Gua
Derivatives
on Template Strand </italic><italic toggle="yes">Utilization</italic>. To evaluate the
utilization
of the plus versus minus strand in the presence of each of
the three exocyclic Gua adducts, we performed experiments
using M13MB102 containing a T positioned opposite the
adduct and probed with an oligonucleotide containing A at
the position equivalent to 6256. Determination of the
percentage of plaques that hybridize with A permitted
estimation of the frequency of utilization of the plus
strand
in vivo (<italic toggle="yes">12</italic>). The plus strand was used as a template
30%
of the time in both LM102 and LM103 strains when no
adducts were present in the plus strand (Table
<xref rid="bi972327rt00004"></xref>). However,
in the presence of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, the minus
strand was used
78% and 82% of the time in LM102 and LM103, respectively. The difference reflects blockage of replication
by
polymerases in the presence of the adduct or replication
following repair of the adduct. Taking these results
into
account, a corrected value of 16% total mutation
frequencies
in the uvrA-deficient strain can be calculated with
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (Table <xref rid="bi972327rt00004"></xref>). The presence of ethanoGua in the vector
led to 51% plus strand use in LM102 and 33% in LM103.
Such observations allow correction of the total
mutation
frequency to 2.2% for ethanoGua in LM103.
HO-ethanoGua
also increased the use of the plus strand (to 72% in
LM102
and 56% in LM103), yielding a corrected total mutation
frequency of ∼0.8% in the uvrA<sup>-</sup> strain.
<table-wrap id="bi972327rt00004" position="float" orientation="portrait"><label>4</label><caption><p>Strand Usage opposite 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua,
HO-ethanoGua, and
EthanoGua in T Constructs<!--%@md;sys;6q@%%@fn;(;vis;full;auto@%+%@fnx;);vis;full@% @sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;T@sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fn;(;vis;full;auto@%−%@fnx;);vis;full@% @sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;G*@sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;%@mx@%[S_EL2;quad]
--></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="5"><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:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="5">% use of plus strand,<italic toggle="yes"><sup>a</sup></italic><sup></sup> base = G*</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">E. coli</italic> strain</oasis:entry><oasis:entry colname="2">Gua</oasis:entry><oasis:entry colname="3">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua</oasis:entry><oasis:entry colname="4">ethanoGua</oasis:entry><oasis:entry colname="5">HO-ethanoGua
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">LM102
</oasis:entry><oasis:entry colname="2">29 ± 6
</oasis:entry><oasis:entry colname="3">78 ± 7
</oasis:entry><oasis:entry colname="4">51 ± 13
</oasis:entry><oasis:entry colname="5">72 ± 8
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">LM103 (uvrA<sup>-)</sup></oasis:entry><oasis:entry colname="2">28 ± 2
</oasis:entry><oasis:entry colname="3">82 ± 5
</oasis:entry><oasis:entry colname="4">33 ± 1
</oasis:entry><oasis:entry colname="5">56 ± 2</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="2"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry namest="1" nameend="4">corrected total mutation frequency (%)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="5"><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:tbody><oasis:row><oasis:entry colname="1">LM103 (uvrA<sup>-)</sup></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">16
</oasis:entry><oasis:entry colname="4">2.2
</oasis:entry><oasis:entry colname="5">0.8</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Results are presented as means ± SD of three
experiments, with
the percentage being determined by dividing the number of
plaques
with DNA hybridized to a<!--%@mt;sys@% %@ex@%32%@exx@%P@sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;%@bold@%A%@rsf@%@sbd;%@kn;1.5q@%@sbd;%@kn;1.5q@%@sbd;%@mx@%
-->probe divided by the total
number of plaques
(<italic toggle="yes">12</italic>).<italic toggle="yes"><sup>b</sup></italic><sup></sup> Results from Table <xref rid="bi972327rt00001"></xref>
are divided by the
fraction of use of the minus strand containing the Gua
derivative.</p></table-wrap-foot></table-wrap></p></sec><sec id="d7e2151"><title>Discussion</title><p>A key result of this study is that 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
and ethanoGua
were both mutagenic in <italic toggle="yes">E. coli</italic>, yielding both
G→A
transitions and G→T tranversions, while HO-ethanoGua was
much less mutagenic. The work clearly establishes that
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, long recognized as a reaction product of Gua
nucleosides or DNA treated with several bifunctional
alkylating agents (<italic toggle="yes">38</italic>), is mutagenic in vivo. All mutations
seen
with 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, ethanoGua, and HO-ethanoGua
were dependent on SOS induction by UV light, suggesting that
polymerase III is involved in adduct bypass and
introduction
of mutations. The mutations seen with
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
ethanoGua were attenuated by the presence of a functional
uvrA gene, indicating the involvement of nucleotide
excision
repair in removal (Table <xref rid="bi972327rt00001"></xref>). The levels of mutations
seen
with HO-ethanoGua were too low to readily determine
whether nucleotide excision repair has a role or not.
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, ethanoGua (Figure <xref rid="bi972327rf00003"></xref>), and HO-ethanoGua (<italic toggle="yes">23</italic>)
pose
strong blocks to replication by several polymerases. A
partial
block also appears to occur in vivo in <italic toggle="yes">E. coli</italic>, as shown
by
the increased replication from the opposite plus strand
(Table
<xref rid="bi972327rt00004"></xref>). These results can be used to normalize mutation
frequencies to reflect the percent of misincorporation
events
occurring as polymerases insert nucleotides opposite the
lesion (in uvrA<sup>-</sup> <italic toggle="yes">E. coli</italic>) (<italic toggle="yes">39</italic>).
The result is a trend of
miscoding in the order 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (16%) >
ethanoGua
(2.2%) > HO-ethanoGua (0.8%) (Table <xref rid="bi972327rt00004"></xref>). Thus, even
though the entire base-pairing region of Gua is blocked,
polymerases still appear to insert C more than 80% of the
time, assuming that the mutation frequencies are not
further
influenced by other DNA repair processes. The
incorporation of any nucleotide, particulary dATP, should not
preserve
the overall geometry of a canonical purine:pyrimidine
pair.
</p><p>The misincorporation patterns seen here with these five-membered exocyclic Gua derivatives can be compared with
those observed with the six-membered homologues (Scheme
<xref rid="bi972327rh00003"></xref>) (<italic toggle="yes">11, 12</italic>). In in vitro studies with
Kf<sup>-</sup>, propanoGua led to
the misincorporation of mainly G and A (<italic toggle="yes">40</italic>), similar to
the
results seen with both 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua with
Kf<sup>-</sup> (pol II<sup>-</sup>, T7<sup>-</sup>, and HIV RT
tended to insert only A)
(<italic toggle="yes">23</italic>). With ethanoGua, the preference for incorporation
by
Kf<sup>-</sup> was G > T ≈ C ≫ A (Figure <xref rid="bi972327rf00003"></xref>; Table <xref rid="bi972327rt00003"></xref>). In
<italic toggle="yes">E. coli</italic>,
both propanoGua and M<sub>1</sub>Gua produced G→A and
G→T
mutations at equal frequencies (<italic toggle="yes">11,
12</italic>), and a similar result
was observed with ethanoGua (Table <xref rid="bi972327rt00001"></xref>). With
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua,
there was a consistently greater fraction of G→A than
G→T
mutations, most readily seen in the absence of uvrA (Table
<xref rid="bi972327rt00002"></xref>). PropanoGua was more mutagenic than M<sub>1</sub>Gua
in
previous studies (<italic toggle="yes">11, 12</italic>), with the total mutation
frequency
rising to ∼39% when corrected for strand use. With
the
five-membered exocyclic rings, the unsaturated adduct
(1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua) was more mutagenic than the saturated analogue
(ethanoGua) (Table <xref rid="bi972327rt00001"></xref>).
</p><p>Differences were seen in the manner in which polymerases
process the five-membered exocyclic adducts. Some
variations were seen when the purified polymerases were used
with the ethanoGua-containing template (Table <xref rid="bi972327rt00003"></xref>), as with
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua (<italic toggle="yes">23</italic>),
although all were
strongly blocked (Figure <xref rid="bi972327rf00003"></xref>). The work presented in
Table
<xref rid="bi972327rt00003"></xref> also reinforces the observation of blockage by the
ethanoGua lesion, although it must be emphasized that the rate-limiting step in these reactions is unknown (under the
conditions used in the assays). The misincorporation
patterns
seen in <italic toggle="yes">E. coli</italic>, where pol III is probably involved, are
quite
different from those observed with pol I (Kf<sup>-</sup>) and pol
II<sup>-</sup>.
Pol III requires the SOS response for all activities
observed
here (Table <xref rid="bi972327rt00001"></xref>). Other DNA adducts have been shown to
yield varying misincorporation results with different polymerases (<italic toggle="yes">23</italic><italic toggle="yes">−</italic><italic toggle="yes">25</italic>).<xref rid="bi972327rX00003" ref-type="bibr"></xref> These
results argue that mutagenesis
should be considered a kinetic process, as opposed to a
thermodynamic process, and that details of mutagenesis
cannot be understood only through structural studies, at
least
in the absence of polymerases.
</p><p>The three five-membered exocyclic rings considered here
appear similar at a casual glance (Scheme <xref rid="bi972327rh00001"></xref>A).
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
was more mutagenic in <italic toggle="yes">E. coli</italic> and also showed a shift
to
G→A transitions, in the absence of uvrA (Table <xref rid="bi972327rt00001"></xref>).
This
might possibly be explained by the increased tendency of
the saturated ethanoGua to pucker. It is possible that
this
minor structural variation is responsible for the
consistently
greater blocking of purified polymerases by ethanoGua >
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (Figure <xref rid="bi972327rf00003"></xref>). Indeed, in most
cases there is very
limited information available regarding
the mechanisms
underlying blockage of polymerases by adducts. HO-ethanoGua has biological properties quite different from
those of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua or ethanoGua. The
saturation of the
ring (<italic toggle="yes">sp</italic><sup>3</sup> binding) cannot be responsible for the
differences
in considering ethanoGua. The low mutation frequency
seen
with HO-ethanoGua is not the result of a strong in vivo
block
to replication, as demonstrated in the results of strand
use
(Table <xref rid="bi972327rt00004"></xref>). One possibility that cannot be excluded is
that
other repair systems, for example, glycosylases, are
highly
active and have not been accounted for in this study.
Bacterial glycosylases have been shown to have
activity
toward some of the ε adducts (<italic toggle="yes">41</italic>). Another
explanation for
the low mutagenicity of HO-ethanoGua is that the structure
exists in multiple forms, as shown previously (Scheme
<xref rid="bi972327rh00005"></xref>)
(<italic toggle="yes">23</italic>). The two epimers at the carbinol exist, as shown
by
CD measurements with the nucleoside (<italic toggle="yes">23</italic>). However,
if
one epimeric form were misincorporating and the other were
not, the mutation frequency would presumably only be
decreased 2-fold. However, the ring-opened form,
<italic toggle="yes">N</italic><sup>2</sup>-(2-oxoethyl)Gua, might be more prominent at the
replication
fork complex and be less mutagenic. A suitable model
for
this might be <italic toggle="yes">N</italic><sup>2</sup>-(2-acetamido)Gua, but to
our knowledge,
no studies have been done with this adduct.
<fig id="bi972327rh00005" position="float" fig-type="scheme" orientation="portrait"><label>5</label><caption><p>Equilibration of Forms of OH-EthanoGua</p></caption><graphic xlink:href="bi972327rh00005.eps" position="float" orientation="portrait"></graphic></fig></p><p>Other stable Gua hemiaminal products include those
derived from glyoxal (<italic toggle="yes">42</italic>), acrolein (<italic toggle="yes">43</italic>), and
crotonaldehyde
and other bifunctional alkylating agents (<italic toggle="yes">44, 45</italic>). The
latter,
HO-propanoGua, is the six-membered ring homologue of
HO-ethanoGua (Scheme <xref rid="bi972327rh00003"></xref>) and has been identified in liver
samples of humans who have not been knowingly exposed
to potential mutagens (<italic toggle="yes">16</italic>). A possible explanation for
its
formation has been proposed, with the adduct arising from
reaction of an epoxide of the lipid peroxidation product
4-hydroxynonenal (<italic toggle="yes">46, 47</italic>). The suitability of
HO-ethanoGua
as a model for these adducts has not been investigated.
However, the synthetic approaches we have utilized here
and
previously (<italic toggle="yes">23</italic>) should be applicable, at least for
HO-propanoGua.
</p><p>The relevance of these studies to the entire issue of
mutagenesis and carcinogenesis caused by vinyl halides and
other compounds is not yet clear. The ε adducts
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Ade, 3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyt,
<italic toggle="yes">N</italic><sup>2</sup>,3-ε-Gua, and
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua have now all
been shown to be mutagenic in various bacterial systems,
and HO-ethanoGua is marginally mutagenic. However,
treatment of isolated DNA with 2-chlorooxirane yields
adducts in the order 1,<italic toggle="yes">N</italic><sup>6</sup>-ε-Ade >
HO-ethanoGua > <italic toggle="yes">N</italic><sup>2</sup>,3-ε-Gua > 3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyt >
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (<italic toggle="yes">22</italic>). Definitive
studies
of the rates of removal of all of these adducts from
mammalian systems have not been completed, and few other
direct comparisons of mutagenicity have been made in
bacterial or mammalian systems.
</p><p>In summary, a total of five structurally related DNA
adducts have now been compared for site-specific mutagenesis at one site in a single vector system, in this and
previous
work (<italic toggle="yes">12, 39</italic>). The significance of this direct
comparison
is emphasized, because assessments of mutagenicity are
often
influenced by sequence contexts and vectors (<italic toggle="yes">8</italic>). A
general
similarity was seen in that the saturated and unsaturated
five-
and six-membered exocyclic ring derivatives are similar,
in
that all are mutagenic and produce G→A transitions and
G→T transversions in vivo (Tables <xref rid="bi972327rt00001"></xref> and <xref rid="bi972327rt00004"></xref>) (<italic toggle="yes">12, 39</italic>).
All
mutations were strongly SOS-dependent, and nucleotide
excision repair was implicated. The presence of a
(hemiaminal) hydroxyl group in the saturated (five-membered)
ring
attenuated the mutagenic response in this system, and if
the
analogy holds, then HO-propanoGua (Scheme <xref rid="bi972327rh00002"></xref>) might be
expected to show low mutagenic acitivity as well.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank L. L. Furge for the polymerases, M.-S. Kim for
helpful suggestions regarding synthesis of modified oligonucleotides, M. D. Cooper and Dr. N. Schnetz-Boutaud for
acquiring some of the NMR spectra, Drs. F. J. Belas and A.
Chaudhary for the mass spectra, Dr. H. J. Einolf for
assistance with CGE and steady-state polymerization
assays,
Dr. N. A. Hosea for her assistance in analysis of steady-state kinetic results, and Dr. K. Teng and D. McCombs for
assistance in preparation of the manuscript.
</p></ack><ref-list><title>References</title><ref id="bi972327rX00001"><element-citation publication-type="journal"><name name-style="western"><surname>Kochetkov</surname><given-names>N. K.</given-names></name><name name-style="western"><surname>Shibaev</surname><given-names>V. N.</given-names></name><name name-style="western"><surname>Kost</surname><given-names>A. A.</given-names></name><source>Tetrahedron Lett.</source><year>1971</year><volume>22</volume><fpage>1993</fpage><lpage>1996</lpage></element-citation></ref><ref id="bi972327rX00002"><element-citation publication-type="journal"><name name-style="western"><surname>Barrio</surname><given-names>J. R.</given-names></name><name name-style="western"><surname>Secrist</surname><given-names>J. A., </given-names><suffix>I</suffix></name><name name-style="western"><surname>Leonard</surname><given-names>N. J.</given-names></name><source>Biochem. Biophys. Res. Commun.</source><year>1972</year><volume>46</volume><fpage>597</fpage><lpage>604</lpage><pub-id pub-id-type="doi">10.1016/S0006-291X(72)80181-5</pub-id></element-citation></ref><ref id="bi972327rX00003"><mixed-citation><name name-style="western"><surname>Singer</surname><given-names>B.</given-names></name>, <name name-style="western"><surname>and Bartsch</surname><given-names>H.</given-names></name> (1986) <italic toggle="yes">The Role of Cyclic Nucleic Acid Adducts </italic><italic toggle="yes">in Carcinogenesis and Mutagenesis</italic>, IARC Scientific Publications, Lyon.</mixed-citation></ref><ref id="bi972327rX00004"><element-citation publication-type="journal"><name name-style="western"><surname>Leonard</surname><given-names>N. J.</given-names></name><source>Chemtracts: Biochem. Mol. Biol.</source><year>1993</year><volume>4</volume><fpage>251</fpage><lpage>284</lpage></element-citation></ref><ref id="bi972327rX00005"><element-citation publication-type="journal"><name name-style="western"><surname>Nakanishi</surname><given-names>K.</given-names></name><name name-style="western"><surname>Furutachi</surname><given-names>N.</given-names></name><name name-style="western"><surname>Funamizu</surname><given-names>M.</given-names></name><name name-style="western"><surname>Grunberger</surname><given-names>D.</given-names></name><name name-style="western"><surname>Weinstein</surname><given-names>I. B.</given-names></name><source>J. Am. Chem. Soc.</source><year>1970</year><volume>92</volume><fpage>7617</fpage><lpage>7619</lpage><pub-id pub-id-type="doi">10.1021/ja00729a035</pub-id></element-citation></ref><ref id="bi972327rX00006"><element-citation publication-type="journal"><name name-style="western"><surname>Basu</surname><given-names>A. K.</given-names></name><name name-style="western"><surname>Wood</surname><given-names>M. L.</given-names></name><name name-style="western"><surname>Niedernhofer</surname><given-names>L. J.</given-names></name><name name-style="western"><surname>Ramos</surname><given-names>L. A.</given-names></name><name name-style="western"><surname>Essigmann</surname><given-names>J. M.</given-names></name><source>Biochemistry</source><year>1993</year><volume>32</volume><fpage>12793</fpage><lpage>12801</lpage></element-citation></ref><ref id="bi972327rX00007"><element-citation publication-type="journal"><name name-style="western"><surname>Palejwala</surname><given-names>V. A.</given-names></name><name name-style="western"><surname>Rzepka</surname><given-names>R. W.</given-names></name><name name-style="western"><surname>Simha</surname><given-names>D.</given-names></name><name name-style="western"><surname>Humayun</surname><given-names>M. Z.</given-names></name><source>Biochemistry</source><year>1993</year><volume>32</volume><fpage>4105</fpage><lpage>4111</lpage></element-citation></ref><ref id="bi972327rX00008"><element-citation publication-type="journal"><name name-style="western"><surname>Moriya</surname><given-names>M.</given-names></name><name name-style="western"><surname>Zhang</surname><given-names>W.</given-names></name><name name-style="western"><surname>Johnson</surname><given-names>F.</given-names></name><name name-style="western"><surname>Grollman</surname><given-names>A. P.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1994</year><volume>91</volume><fpage>11899</fpage><lpage>11903</lpage><pub-id pub-id-type="doi">10.1073/pnas.91.25.11899</pub-id></element-citation></ref><ref id="bi972327rX00009"><element-citation publication-type="journal"><name name-style="western"><surname>Cheng</surname><given-names>K. C.</given-names></name><name name-style="western"><surname>Preston</surname><given-names>B. D.</given-names></name><name name-style="western"><surname>Cahill</surname><given-names>D. S.</given-names></name><name name-style="western"><surname>Dosanjh</surname><given-names>M. K.</given-names></name><name name-style="western"><surname>Singer</surname><given-names>B.</given-names></name><name name-style="western"><surname>Loeb</surname><given-names>L. A.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1991</year><volume>88</volume><fpage>9974</fpage><lpage>9978</lpage><pub-id pub-id-type="doi">10.1073/pnas.88.22.9974</pub-id></element-citation></ref><ref id="bi972327rX00010"><element-citation publication-type="journal"><name name-style="western"><surname>Shibutani</surname><given-names>S.</given-names></name><name name-style="western"><surname>Suzuki</surname><given-names>N.</given-names></name><name name-style="western"><surname>Matsumoto</surname><given-names>Y.</given-names></name><name name-style="western"><surname>Grollman</surname><given-names>A. P.</given-names></name><source>Biochemistry</source><year>1996</year><volume>35</volume><fpage>14992</fpage><lpage>14998</lpage></element-citation></ref><ref id="bi972327rX00011"><element-citation publication-type="journal"><name name-style="western"><surname>Burcham</surname><given-names>P. C.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>J. Biol. Chem.</source><year>1994</year><volume>269</volume><fpage>28844</fpage><lpage>28850</lpage></element-citation></ref><ref id="bi972327rX00012"><element-citation publication-type="journal"><name name-style="western"><surname>Fink</surname><given-names>S. P.</given-names></name><name name-style="western"><surname>Reddy</surname><given-names>G. R.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1997</year><volume>94</volume><fpage>8652</fpage><lpage>8657</lpage><pub-id pub-id-type="doi">10.1073/pnas.94.16.8652</pub-id></element-citation></ref><ref id="bi972327rX00013"><element-citation publication-type="journal"><name name-style="western"><surname>Fedtke</surname><given-names>N.</given-names></name><name name-style="western"><surname>Boucheron</surname><given-names>J. A.</given-names></name><name name-style="western"><surname>Walker</surname><given-names>V. E.</given-names></name><name name-style="western"><surname>Swenberg</surname><given-names>J. A.</given-names></name><source>Carcinogenesis</source><year>1990</year><volume>11</volume><fpage>1287</fpage><lpage>1292</lpage></element-citation></ref><ref id="bi972327rX00014"><element-citation publication-type="journal"><name name-style="western"><surname>Kronberg</surname><given-names>L.</given-names></name><name name-style="western"><surname>Sjöholm</surname><given-names>R.</given-names></name><name name-style="western"><surname>Karlsson</surname><given-names>S.</given-names></name><source>Chem. Res. Toxicol.</source><year>1992</year><volume>5</volume><fpage>852</fpage><lpage>855</lpage><pub-id pub-id-type="doi">10.1021/tx00030a019</pub-id></element-citation></ref><ref id="bi972327rX00015"><element-citation publication-type="journal"><name name-style="western"><surname>Chaudhary</surname><given-names>A. K.</given-names></name><name name-style="western"><surname>Nokubo</surname><given-names>M.</given-names></name><name name-style="western"><surname>Reddy</surname><given-names>G. R.</given-names></name><name name-style="western"><surname>Yeola</surname><given-names>S. N.</given-names></name><name name-style="western"><surname>Morrow</surname><given-names>J. D.</given-names></name><name name-style="western"><surname>Blair</surname><given-names>I. A.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>Science</source><year>1994</year><volume>265</volume><fpage>1580</fpage><lpage>1582</lpage></element-citation></ref><ref id="bi972327rX00016"><element-citation publication-type="journal"><name name-style="western"><surname>Nath</surname><given-names>R. G.</given-names></name><name name-style="western"><surname>Chung</surname><given-names>F. L.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1994</year><volume>91</volume><fpage>7491</fpage><lpage>7495</lpage><pub-id pub-id-type="doi">10.1073/pnas.91.16.7491</pub-id></element-citation></ref><ref id="bi972327rX00017"><element-citation publication-type="journal"><name name-style="western"><surname>Rouzer</surname><given-names>C. A.</given-names></name><name name-style="western"><surname>Chaudhary</surname><given-names>A. K.</given-names></name><name name-style="western"><surname>Nokubo</surname><given-names>M.</given-names></name><name name-style="western"><surname>Ferguson</surname><given-names>D. M.</given-names></name><name name-style="western"><surname>Reddy</surname><given-names>G. R.</given-names></name><name name-style="western"><surname>Blair</surname><given-names>I. A.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>Chem. Res. Toxicol.</source><year>1997</year><volume>10</volume><fpage>181</fpage><lpage>188</lpage><pub-id pub-id-type="doi">10.1021/tx9601216</pub-id></element-citation></ref><ref id="bi972327rX00018"><element-citation publication-type="journal"><name name-style="western"><surname>Nair</surname><given-names>J.</given-names></name><name name-style="western"><surname>Barbin</surname><given-names>A.</given-names></name><name name-style="western"><surname>Guichard</surname><given-names>Y.</given-names></name><name name-style="western"><surname>Bartsch</surname><given-names>H.</given-names></name><source>Carcinogenesis</source><year>1995</year><volume>16</volume><fpage>613</fpage><lpage>617</lpage></element-citation></ref><ref id="bi972327rX00019"><element-citation publication-type="journal"><name name-style="western"><surname>Ames</surname><given-names>B. N.</given-names></name><name name-style="western"><surname>Gold</surname><given-names>L. S.</given-names></name><source>Mutat. Res.</source><year>1991</year><volume>250</volume><fpage>3</fpage><lpage>16</lpage></element-citation></ref><ref id="bi972327rX00020"><element-citation publication-type="journal"><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><name name-style="western"><surname>Burcham</surname><given-names>P. C.</given-names></name><source>Chem. Res. Toxicol.</source><year>1993</year><volume>6</volume><fpage>771</fpage><lpage>785</lpage><pub-id pub-id-type="doi">10.1021/tx00036a005</pub-id></element-citation></ref><ref id="bi972327rX00021"><element-citation publication-type="journal"><name name-style="western"><surname>Leonard</surname><given-names>N. J.</given-names></name><source>Crit. Rev. Biochem.</source><year>1984</year><volume>15</volume><fpage>125</fpage><lpage>199</lpage><pub-id pub-id-type="doi">10.3109/10409238409102299</pub-id></element-citation></ref><ref id="bi972327rX00022"><element-citation publication-type="journal"><name name-style="western"><surname>Müller</surname><given-names>M.</given-names></name><name name-style="western"><surname>Belas</surname><given-names>F. J.</given-names></name><name name-style="western"><surname>Blair</surname><given-names>I. A.</given-names></name><name name-style="western"><surname>Guengerich</surname><given-names>F. P.</given-names></name><source>Chem. Res. Toxicol.</source><year>1997</year><volume>10</volume><fpage>242</fpage><lpage>247</lpage><pub-id pub-id-type="doi">10.1021/tx960124i</pub-id></element-citation></ref><ref id="bi972327rX00023"><element-citation publication-type="journal"><name name-style="western"><surname>Langouët</surname><given-names>S.</given-names></name><name name-style="western"><surname>Müller</surname><given-names>M.</given-names></name><name name-style="western"><surname>Guengerich</surname><given-names>F. P.</given-names></name><source>Biochemistry</source><year>1997</year><volume>36</volume><fpage>6069</fpage><lpage>6079</lpage></element-citation></ref><ref id="bi972327rX00024"><element-citation publication-type="journal"><name name-style="western"><surname>Lowe</surname><given-names>L. G.</given-names></name><name name-style="western"><surname>Guengerich</surname><given-names>F. P.</given-names></name><source>Biochemistry</source><year>1996</year><volume>35</volume><fpage>9840</fpage><lpage>9849</lpage></element-citation></ref><ref id="bi972327rX00025"><element-citation publication-type="journal"><name name-style="western"><surname>Furge</surname><given-names>L. L.</given-names></name><name name-style="western"><surname>Guengerich</surname><given-names>F. P.</given-names></name><source>Biochemistry</source><year>1997</year><volume>36</volume><fpage>6475</fpage><lpage>6487</lpage></element-citation></ref><ref id="bi972327rX00026"><element-citation publication-type="journal"><name name-style="western"><surname>Marinelli</surname><given-names>E. R.</given-names></name><name name-style="western"><surname>Johnson</surname><given-names>F.</given-names></name><name name-style="western"><surname>Iden</surname><given-names>C. R.</given-names></name><name name-style="western"><surname>Yu</surname><given-names>P. L.</given-names></name><source>Chem. Res. Toxicol.</source><year>1990</year><volume>3</volume><fpage>49</fpage><lpage>58</lpage><pub-id pub-id-type="doi">10.1021/tx00013a009</pub-id></element-citation></ref><ref id="bi972327rX00027"><element-citation publication-type="journal"><name name-style="western"><surname>Decorte</surname><given-names>B. L.</given-names></name><name name-style="western"><surname>Tsarouhtsis</surname><given-names>D.</given-names></name><name name-style="western"><surname>Kuchimanchi</surname><given-names>S.</given-names></name><name name-style="western"><surname>Cooper</surname><given-names>M. D.</given-names></name><name name-style="western"><surname>Horton</surname><given-names>P.</given-names></name><name name-style="western"><surname>Harris</surname><given-names>C. M.</given-names></name><name name-style="western"><surname>Harris</surname><given-names>T. M.</given-names></name><source>Chem. Res. Toxicol.</source><year>1996</year><volume>9</volume><fpage>630</fpage><lpage>637</lpage><pub-id pub-id-type="doi">10.1021/tx9501795</pub-id></element-citation></ref><ref id="bi972327rX00028"><mixed-citation><name name-style="western"><surname>Borer</surname><given-names>P. N.</given-names></name> (1975) in <italic toggle="yes">Handbook of Biochemistry and Molecular Biology</italic> (<name name-style="western"><surname>Fasman</surname><given-names>G. D.</given-names></name>, Ed.)3rd ed., pp 589−580, CRC Press, Cleveland, OH.</mixed-citation></ref><ref id="bi972327rX00029"><element-citation publication-type="journal"><name name-style="western"><surname>Benamira</surname><given-names>M.</given-names></name><name name-style="western"><surname>Singh</surname><given-names>U.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>J. Biol. Chem.</source><year>1992</year><volume>267</volume><fpage>22392</fpage><lpage>22400</lpage></element-citation></ref><ref id="bi972327rX00030"><element-citation publication-type="journal"><name name-style="western"><surname>Benamira</surname><given-names>M.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>Chem. Res. Toxicol.</source><year>1993</year><volume>6</volume><fpage>317</fpage><lpage>327</lpage><pub-id pub-id-type="doi">10.1021/tx00033a011</pub-id></element-citation></ref><ref id="bi972327rX00031"><mixed-citation><name name-style="western"><surname>Sambrook</surname><given-names>J.</given-names></name>, <name name-style="western"><surname>Fritsch</surname><given-names>E. F.</given-names></name>, <name name-style="western"><surname>and Maniatis</surname><given-names>T.</given-names></name> (1989) <italic toggle="yes">Molecular Cloning: </italic><italic toggle="yes">A Laboratory Manual</italic>, p A.1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</mixed-citation></ref><ref id="bi972327rX00032"><mixed-citation><name name-style="western"><surname>Sambrook</surname><given-names>J.</given-names></name>, <name name-style="western"><surname>Fritsch</surname><given-names>E. F.</given-names></name>, <name name-style="western"><surname>and Maniatis</surname><given-names>T.</given-names></name> (1989) <italic toggle="yes">Molecular Cloning: </italic><italic toggle="yes">A Laboratory Manual</italic>, p B.15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</mixed-citation></ref><ref id="bi972327rX00033"><element-citation publication-type="journal"><name name-style="western"><surname>Fink</surname><given-names>S. P.</given-names></name><name name-style="western"><surname>Reddy</surname><given-names>G. R.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>Chem. Res. Toxicol.</source><year>1996</year><volume>9</volume><fpage>277</fpage><lpage>283</lpage><pub-id pub-id-type="doi">10.1021/tx950060w</pub-id></element-citation></ref><ref id="bi972327rX00034"><element-citation publication-type="journal"><name name-style="western"><surname>Boosalis</surname><given-names>M. S.</given-names></name><name name-style="western"><surname>Petruska</surname><given-names>J.</given-names></name><name name-style="western"><surname>Goodman</surname><given-names>M. F.</given-names></name><source>J. Biol. Chem.</source><year>1987</year><volume>262</volume><fpage>14689</fpage><lpage>14696</lpage></element-citation></ref><ref id="bi972327rX00035"><element-citation publication-type="journal"><name name-style="western"><surname>Maxam</surname><given-names>A. M.</given-names></name><name name-style="western"><surname>Gilbert</surname><given-names>W.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1977</year><volume>74</volume><fpage>560</fpage><lpage>564</lpage><pub-id pub-id-type="doi">10.1073/pnas.74.2.560</pub-id></element-citation></ref><ref id="bi972327rX00036"><element-citation publication-type="journal"><name name-style="western"><surname>Maxam</surname><given-names>A. M.</given-names></name><name name-style="western"><surname>Gilbert</surname><given-names>W.</given-names></name><source>Methods Enzymol.</source><year>1980</year><volume>65</volume><fpage>499</fpage><lpage>560</lpage></element-citation></ref><ref id="bi972327rX00037"><element-citation publication-type="journal"><name name-style="western"><surname>Friedman</surname><given-names>T.</given-names></name><name name-style="western"><surname>Brown</surname><given-names>D. H.</given-names></name><source>Nucleic Acids Res.</source><year>1978</year><volume>5</volume><fpage>615</fpage><lpage>622</lpage></element-citation></ref><ref id="bi972327rX00038"><element-citation publication-type="journal"><name name-style="western"><surname>Sattsangi</surname><given-names>P. D.</given-names></name><name name-style="western"><surname>Leonard</surname><given-names>N. J.</given-names></name><name name-style="western"><surname>Frihart</surname><given-names>C. R.</given-names></name><source>J. Org. Chem.</source><year>1977</year><volume>42</volume><fpage>3292</fpage><lpage>3296</lpage><pub-id pub-id-type="doi">10.1021/jo00440a020</pub-id></element-citation></ref><ref id="bi972327rX00039"><element-citation publication-type="journal"><name name-style="western"><surname>Johnson</surname><given-names>K. A.</given-names></name><name name-style="western"><surname>Fink</surname><given-names>S. P.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>J. Biol. Chem.</source><year>1997</year><volume>272</volume><fpage>11434</fpage><lpage>11438</lpage><pub-id pub-id-type="doi">10.1074/jbc.272.17.11434</pub-id></element-citation></ref><ref id="bi972327rX00040"><element-citation publication-type="journal"><name name-style="western"><surname>Hashim</surname><given-names>M.</given-names></name><name name-style="western"><surname>Marnett</surname><given-names>L. J.</given-names></name><source>J. Biol. Chem.</source><year>1996</year><volume>271</volume><fpage>9160</fpage><lpage>9165</lpage><pub-id pub-id-type="doi">10.1074/jbc.271.15.9160</pub-id></element-citation></ref><ref id="bi972327rX00041"><element-citation publication-type="journal"><name name-style="western"><surname>Dosanjh</surname><given-names>M. K.</given-names></name><name name-style="western"><surname>Chenna</surname><given-names>A.</given-names></name><name name-style="western"><surname>Kim</surname><given-names>E.</given-names></name><name name-style="western"><surname>Fraenkel-Conrat</surname><given-names>H.</given-names></name><name name-style="western"><surname>Samson</surname><given-names>L.</given-names></name><name name-style="western"><surname>Singer</surname><given-names>B.</given-names></name><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>1994</year><volume>91</volume><fpage>1024</fpage><lpage>1028</lpage><pub-id pub-id-type="doi">10.1073/pnas.91.3.1024</pub-id></element-citation></ref><ref id="bi972327rX00042"><element-citation publication-type="journal"><name name-style="western"><surname>Shapiro</surname><given-names>R.</given-names></name><name name-style="western"><surname>Cohen</surname><given-names>B. I.</given-names></name><name name-style="western"><surname>Shiuey</surname><given-names>S. J.</given-names></name><name name-style="western"><surname>Maurer</surname><given-names>H.</given-names></name><source>Biochemistry</source><year>1969</year><volume>8</volume><fpage>238</fpage><lpage>245</lpage></element-citation></ref><ref id="bi972327rX00043"><element-citation publication-type="journal"><name name-style="western"><surname>Foiles</surname><given-names>P. G.</given-names></name><name name-style="western"><surname>Akerkar</surname><given-names>S. A.</given-names></name><name name-style="western"><surname>Miglietta</surname><given-names>L. M.</given-names></name><name name-style="western"><surname>Chung</surname><given-names>F. L.</given-names></name><source>Carcinogenesis</source><year>1990</year><volume>11</volume><fpage>2059</fpage><lpage>2061</lpage></element-citation></ref><ref id="bi972327rX00044"><element-citation publication-type="journal"><name name-style="western"><surname>Chung</surname><given-names>F. L.</given-names></name><name name-style="western"><surname>Krzeminski</surname><given-names>J.</given-names></name><name name-style="western"><surname>Wang</surname><given-names>M.</given-names></name><name name-style="western"><surname>Chen</surname><given-names>H. J. C.</given-names></name><name name-style="western"><surname>Prokopczyk</surname><given-names>B.</given-names></name><source>Chem. Res. Toxicol.</source><year>1994</year><volume>7</volume><fpage>62</fpage><lpage>67</lpage><pub-id pub-id-type="doi">10.1021/tx00037a009</pub-id></element-citation></ref><ref id="bi972327rX00045"><element-citation publication-type="journal"><name name-style="western"><surname>Chung</surname><given-names>F.-L.</given-names></name><name name-style="western"><surname>Hecht</surname><given-names>S. S.</given-names></name><source>Cancer Res.</source><year>1983</year><volume>43</volume><fpage>1230</fpage><lpage>1235</lpage></element-citation></ref><ref id="bi972327rX00046"><element-citation publication-type="journal"><name name-style="western"><surname>Chung</surname><given-names>F. L.</given-names></name><name name-style="western"><surname>Chen</surname><given-names>H. J. C.</given-names></name><name name-style="western"><surname>Guttenplan</surname><given-names>J. B.</given-names></name><name name-style="western"><surname>Nishikawa</surname><given-names>A.</given-names></name><name name-style="western"><surname>Hard</surname><given-names>G. C.</given-names></name><source>Carcinogenesis</source><year>1993</year><volume>14</volume><fpage>2073</fpage><lpage>2077</lpage></element-citation></ref><ref id="bi972327rX00047"><element-citation publication-type="journal"><name name-style="western"><surname>El Ghissassi</surname><given-names>F.</given-names></name><name name-style="western"><surname>Barbin</surname><given-names>A.</given-names></name><name name-style="western"><surname>Nair</surname><given-names>J.</given-names></name><name name-style="western"><surname>Bartsch</surname><given-names>H.</given-names></name><source>Chem. Res. Toxicol.</source><year>1995</year><volume>8</volume><fpage>278</fpage><lpage>283</lpage><pub-id pub-id-type="doi">10.1021/tx00044a013</pub-id></element-citation></ref><ref id="bi972327rb00001"><mixed-citation><comment>Abbreviations: ε, etheno; Gua or G, guanine; 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, 1,<italic toggle="yes">N</italic><sup>2</sup>-ethenoguanine (or 9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine); ethanoGua, 1,<italic toggle="yes">N</italic><sup>2</sup>-ethanoguanine (5,6,7,9-tetrahydro-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine); HO-ethanoGua, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazopurine; dGuo, deoxyguanosine; ethanodGuo, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine 2-deoxyribosyl nucleoside; M<sub>1</sub>Gua, pyrimido[1,2-<italic toggle="yes">a</italic>]purin-10(3<italic toggle="yes">H</italic>)one; propanoGua, 1,<italic toggle="yes">N</italic><sup>2</sup>-propanoguanine; propanodGuo, 1,<italic toggle="yes">N</italic><sup>2</sup>-propano-2‘-deoxyguanosine; HO-propanoGua, 8-hydroxypropanoGua; CGE, capillary gel electrophoresis; MOPS, 3-(<italic toggle="yes">N</italic>-morphilino)propanesulfonic acid; SSC, saline sodium citrate buffer (15 mM sodium citrate buffer (pH 7.0) containing 150 mM NaCl); SSPE, 10 sodium phosphate buffer (pH 7.4) containing 1.0 mM EDTA and 150 mM NaCl; Kf<sup>-</sup>, <italic toggle="yes">E. coli</italic> polymerase I (Klenow fragment) exo<sup>-</sup>; pol II<sup>-</sup>, <italic toggle="yes">E. coli</italic> polymerase II exo<sup>-</sup>; T7<sup>-</sup>, bacteriophage T7 polymerase exo<sup>-</sup>/thioredoxin complex; HIV RT, human immunodeficiency virus-1 reverse transcriptase; ES MS, electrospray mass spectrometry; Tris, tris(hydroxymethyl)aminomethane; EDTA, (ethylenedinitrilo)acetic acid.</comment></mixed-citation></ref><ref id="bi972327rb00002"><mixed-citation><comment>Some degradation of the primer was observed with HIV RT, when the template contained either 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua or ethanoGua (Figure <xref rid="bi972327rf00003"></xref>D). The basis of this is unknown, but similar results have been seen with other blocking lesions [<italic toggle="yes">23</italic>; M.-S. Kim and F. P. Guengerich (<italic toggle="yes">Chem. Res. Toxicol.</italic>, in press)] and with several commercial preparations of HIV RT (with M<sub>1</sub>Gua).</comment></mixed-citation></ref><ref id="bi972327rb00003"><mixed-citation><comment>M. S. Kim and F. P. Guengerich (<italic toggle="yes">Chem. Res. Toxicol.</italic>, in press).</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†</title></titleInfo><titleInfo contentType="CDATA"><title>Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†</title></titleInfo><name type="personal"><namePart type="family">LANGOUëT</namePart><namePart type="given">Sophie</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</affiliation><affiliation> Current address: INSERM U456, Faculté desSciences Pharmaceutiques et Biologiques 2, Avenue du Professeur Léon Bernard,35043Rennes cedex, France.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MICAN</namePart><namePart type="given">Adrienne N.</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MüLLER</namePart><namePart type="given">Michael</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</affiliation><affiliation> Current address: Abteilung Arbeits- undSozialmedizin der Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">FINK</namePart><namePart type="given">Stephen P.</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">Lawrence J.</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MUHLE</namePart><namePart type="given">Steven A.</namePart><affiliation>Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0146</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 of Medicine,Nashville, Tennessee 37232-0146</affiliation><affiliation> Address correspondence to this author: Department ofBiochemistry, Vanderbilt University School of Medicine,638Medical Research Building I, Nashville, TN 37232-0146.Telephone: (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">1998-03-26</dateCreated><dateIssued encoding="w3cdtf">1998-04-14</dateIssued><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><note type="footnote" ID="bi972327rAF2"> This research was supported in part by United States Public Health Service Grants R35 CA44353 (F.P.G.), R35 CA47479 (L.J.M.), P30 ES00267 (F.P.G., L.J.M.), T32 ES07028 (A.N.M.), and T32 GM07347 (S.A.M.) and a fellowship from the Deutsche Forschungsgemeinschaft (M.M.).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1,N2-Ethenoguanine (1,N2-ε-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G→A, 0.74% G→T, and 0.09% G→C changes found in uvrA- bacteria. 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-ε-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007−0.19% for G→A, C, or T changes. The saturated etheno ring derivative of 1,N2-ε-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G→A and G→T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-ε-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.</abstract><note type="footnote" ID="bi972327rAF2"> This research was supported in part by United States Public Health Service Grants R35 CA44353 (F.P.G.), R35 CA47479 (L.J.M.), P30 ES00267 (F.P.G., L.J.M.), T32 ES07028 (A.N.M.), and T32 GM07347 (S.A.M.) and a fellowship from the Deutsche Forschungsgemeinschaft (M.M.).</note><relatedItem type="host"><titleInfo><title>Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>Biochemistry</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><identifier type="ISSN">0006-2960</identifier><identifier type="eISSN">1520-4995</identifier><identifier type="acspubs">bi</identifier><identifier type="coden">BICHAW</identifier><identifier type="uri">pubs.acs.org/biochemistry</identifier><part><date>1998</date><detail type="volume"><caption>vol.</caption><number>37</number></detail><detail type="issue"><caption>no.</caption><number>15</number></detail><extent unit="pages"><start>5184</start><end>5193</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00001" displayLabel="bibbi972327rX00001"><name type="personal"><namePart type="family">KOCHETKOV</namePart><namePart type="given">N. K.</namePart></name><name type="personal"><namePart type="family">SHIBAEV</namePart><namePart type="given">V. N.</namePart></name><name type="personal"><namePart type="family">KOST</namePart><namePart type="given">A. A.</namePart></name><titleInfo><title>Tetrahedron Lett.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Kochetkov N. K. Shibaev V. N. Kost A. A. Tetrahedron Lett. 1971 22 1993 1996</note><part><date>1971</date><detail type="volume"><caption>vol.</caption><number>22</number></detail><extent unit="pages"><start>1993</start><end>1996</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00002" displayLabel="bibbi972327rX00002"><name type="personal"><namePart type="family">BARRIO</namePart><namePart type="given">J. R.</namePart></name><name type="personal"><namePart type="family">SECRIST</namePart><namePart type="given">J. A.,</namePart></name><name type="personal"><namePart type="family">LEONARD</namePart><namePart type="given">N. J.</namePart></name><titleInfo><title>Biochem. Biophys. Res. Commun.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Barrio J. R. Secrist J. A., I Leonard N. J. Biochem. Biophys. Res. Commun. 1972 46 597 604 10.1016/S0006-291X(72)80181-5</note><identifier type="doi">10.1016/S0006-291X(72)80181-5</identifier><part><date>1972</date><detail type="volume"><caption>vol.</caption><number>46</number></detail><extent unit="pages"><start>597</start><end>604</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00003" displayLabel="bibbi972327rX00003"><name type="personal"><namePart type="family">SINGER</namePart><namePart type="given">B.</namePart></name><name type="personal"><namePart type="family">AND BARTSCH</namePart><namePart type="given">H.</namePart></name><titleInfo><title>The Role of Cyclic Nucleic Acid Adducts </title></titleInfo><note type="content-in-line">SingerB., and BartschH. (1986) The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis, IARC Scientific Publications, Lyon.</note></relatedItem><relatedItem type="references" ID="bi972327rX00004" displayLabel="bibbi972327rX00004"><name type="personal"><namePart type="family">LEONARD</namePart><namePart type="given">N. J.</namePart></name><titleInfo><title>Chemtracts: Biochem. Mol. Biol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Leonard N. J. Chemtracts: Biochem. Mol. Biol. 1993 4 251 284</note><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>4</number></detail><extent unit="pages"><start>251</start><end>284</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00005" displayLabel="bibbi972327rX00005"><name type="personal"><namePart type="family">NAKANISHI</namePart><namePart type="given">K.</namePart></name><name type="personal"><namePart type="family">FURUTACHI</namePart><namePart type="given">N.</namePart></name><name type="personal"><namePart type="family">FUNAMIZU</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">GRUNBERGER</namePart><namePart type="given">D.</namePart></name><name type="personal"><namePart type="family">WEINSTEIN</namePart><namePart type="given">I. B.</namePart></name><titleInfo><title>J. Am. Chem. Soc.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Nakanishi K. Furutachi N. Funamizu M. Grunberger D. Weinstein I. B. J. Am. Chem. Soc. 1970 92 7617 7619 10.1021/ja00729a035</note><identifier type="doi">10.1021/ja00729a035</identifier><part><date>1970</date><detail type="volume"><caption>vol.</caption><number>92</number></detail><extent unit="pages"><start>7617</start><end>7619</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00006" displayLabel="bibbi972327rX00006"><name type="personal"><namePart type="family">BASU</namePart><namePart type="given">A. K.</namePart></name><name type="personal"><namePart type="family">WOOD</namePart><namePart type="given">M. L.</namePart></name><name type="personal"><namePart type="family">NIEDERNHOFER</namePart><namePart type="given">L. J.</namePart></name><name type="personal"><namePart type="family">RAMOS</namePart><namePart type="given">L. A.</namePart></name><name type="personal"><namePart type="family">ESSIGMANN</namePart><namePart type="given">J. M.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Basu A. K. Wood M. L. Niedernhofer L. J. Ramos L. A. Essigmann J. M. Biochemistry 1993 32 12793 12801</note><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>32</number></detail><extent unit="pages"><start>12793</start><end>12801</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00007" displayLabel="bibbi972327rX00007"><name type="personal"><namePart type="family">PALEJWALA</namePart><namePart type="given">V. A.</namePart></name><name type="personal"><namePart type="family">RZEPKA</namePart><namePart type="given">R. W.</namePart></name><name type="personal"><namePart type="family">SIMHA</namePart><namePart type="given">D.</namePart></name><name type="personal"><namePart type="family">HUMAYUN</namePart><namePart type="given">M. Z.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Palejwala V. A. Rzepka R. W. Simha D. Humayun M. Z. Biochemistry 1993 32 4105 4111</note><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>32</number></detail><extent unit="pages"><start>4105</start><end>4111</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00008" displayLabel="bibbi972327rX00008"><name type="personal"><namePart type="family">MORIYA</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">ZHANG</namePart><namePart type="given">W.</namePart></name><name type="personal"><namePart type="family">JOHNSON</namePart><namePart type="given">F.</namePart></name><name type="personal"><namePart type="family">GROLLMAN</namePart><namePart type="given">A. P.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Moriya M. Zhang W. Johnson F. Grollman A. P. Proc. Natl. Acad. Sci. U.S.A. 1994 91 11899 11903 10.1073/pnas.91.25.11899</note><identifier type="doi">10.1073/pnas.91.25.11899</identifier><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>91</number></detail><extent unit="pages"><start>11899</start><end>11903</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00009" displayLabel="bibbi972327rX00009"><name type="personal"><namePart type="family">CHENG</namePart><namePart type="given">K. C.</namePart></name><name type="personal"><namePart type="family">PRESTON</namePart><namePart type="given">B. D.</namePart></name><name type="personal"><namePart type="family">CAHILL</namePart><namePart type="given">D. S.</namePart></name><name type="personal"><namePart type="family">DOSANJH</namePart><namePart type="given">M. K.</namePart></name><name type="personal"><namePart type="family">SINGER</namePart><namePart type="given">B.</namePart></name><name type="personal"><namePart type="family">LOEB</namePart><namePart type="given">L. A.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Cheng K. C. Preston B. D. Cahill D. S. Dosanjh M. K. Singer B. Loeb L. A. Proc. Natl. Acad. Sci. U.S.A. 1991 88 9974 9978 10.1073/pnas.88.22.9974</note><identifier type="doi">10.1073/pnas.88.22.9974</identifier><part><date>1991</date><detail type="volume"><caption>vol.</caption><number>88</number></detail><extent unit="pages"><start>9974</start><end>9978</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00010" displayLabel="bibbi972327rX00010"><name type="personal"><namePart type="family">SHIBUTANI</namePart><namePart type="given">S.</namePart></name><name type="personal"><namePart type="family">SUZUKI</namePart><namePart type="given">N.</namePart></name><name type="personal"><namePart type="family">MATSUMOTO</namePart><namePart type="given">Y.</namePart></name><name type="personal"><namePart type="family">GROLLMAN</namePart><namePart type="given">A. P.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Shibutani S. Suzuki N. Matsumoto Y. Grollman A. P. Biochemistry 1996 35 14992 14998</note><part><date>1996</date><detail type="volume"><caption>vol.</caption><number>35</number></detail><extent unit="pages"><start>14992</start><end>14998</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00011" displayLabel="bibbi972327rX00011"><name type="personal"><namePart type="family">BURCHAM</namePart><namePart type="given">P. C.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>J. Biol. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Burcham P. C. Marnett L. J. J. Biol. Chem. 1994 269 28844 28850</note><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>269</number></detail><extent unit="pages"><start>28844</start><end>28850</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00012" displayLabel="bibbi972327rX00012"><name type="personal"><namePart type="family">FINK</namePart><namePart type="given">S. P.</namePart></name><name type="personal"><namePart type="family">REDDY</namePart><namePart type="given">G. R.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Fink S. P. Reddy G. R. Marnett L. J. Proc. Natl. Acad. Sci. U.S.A. 1997 94 8652 8657 10.1073/pnas.94.16.8652</note><identifier type="doi">10.1073/pnas.94.16.8652</identifier><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>94</number></detail><extent unit="pages"><start>8652</start><end>8657</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00013" displayLabel="bibbi972327rX00013"><name type="personal"><namePart type="family">FEDTKE</namePart><namePart type="given">N.</namePart></name><name type="personal"><namePart type="family">BOUCHERON</namePart><namePart type="given">J. A.</namePart></name><name type="personal"><namePart type="family">WALKER</namePart><namePart type="given">V. E.</namePart></name><name type="personal"><namePart type="family">SWENBERG</namePart><namePart type="given">J. A.</namePart></name><titleInfo><title>Carcinogenesis</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Fedtke N. Boucheron J. A. Walker V. E. Swenberg J. A. Carcinogenesis 1990 11 1287 1292</note><part><date>1990</date><detail type="volume"><caption>vol.</caption><number>11</number></detail><extent unit="pages"><start>1287</start><end>1292</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00014" displayLabel="bibbi972327rX00014"><name type="personal"><namePart type="family">KRONBERG</namePart><namePart type="given">L.</namePart></name><name type="personal"><namePart type="family">SJöHOLM</namePart><namePart type="given">R.</namePart></name><name type="personal"><namePart type="family">KARLSSON</namePart><namePart type="given">S.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Kronberg L. Sjöholm R. Karlsson S. Chem. Res. Toxicol. 1992 5 852 855 10.1021/tx00030a019</note><identifier type="doi">10.1021/tx00030a019</identifier><part><date>1992</date><detail type="volume"><caption>vol.</caption><number>5</number></detail><extent unit="pages"><start>852</start><end>855</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00015" displayLabel="bibbi972327rX00015"><name type="personal"><namePart type="family">CHAUDHARY</namePart><namePart type="given">A. K.</namePart></name><name type="personal"><namePart type="family">NOKUBO</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">REDDY</namePart><namePart type="given">G. R.</namePart></name><name type="personal"><namePart type="family">YEOLA</namePart><namePart type="given">S. N.</namePart></name><name type="personal"><namePart type="family">MORROW</namePart><namePart type="given">J. D.</namePart></name><name type="personal"><namePart type="family">BLAIR</namePart><namePart type="given">I. A.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>Science</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Chaudhary A. K. Nokubo M. Reddy G. R. Yeola S. N. Morrow J. D. Blair I. A. Marnett L. J. Science 1994 265 1580 1582</note><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>265</number></detail><extent unit="pages"><start>1580</start><end>1582</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00016" displayLabel="bibbi972327rX00016"><name type="personal"><namePart type="family">NATH</namePart><namePart type="given">R. G.</namePart></name><name type="personal"><namePart type="family">CHUNG</namePart><namePart type="given">F. L.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Nath R. G. Chung F. L. Proc. Natl. Acad. Sci. U.S.A. 1994 91 7491 7495 10.1073/pnas.91.16.7491</note><identifier type="doi">10.1073/pnas.91.16.7491</identifier><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>91</number></detail><extent unit="pages"><start>7491</start><end>7495</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00017" displayLabel="bibbi972327rX00017"><name type="personal"><namePart type="family">ROUZER</namePart><namePart type="given">C. A.</namePart></name><name type="personal"><namePart type="family">CHAUDHARY</namePart><namePart type="given">A. K.</namePart></name><name type="personal"><namePart type="family">NOKUBO</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">FERGUSON</namePart><namePart type="given">D. M.</namePart></name><name type="personal"><namePart type="family">REDDY</namePart><namePart type="given">G. R.</namePart></name><name type="personal"><namePart type="family">BLAIR</namePart><namePart type="given">I. A.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Rouzer C. A. Chaudhary A. K. Nokubo M. Ferguson D. M. Reddy G. R. Blair I. A. Marnett L. J. Chem. Res. Toxicol. 1997 10 181 188 10.1021/tx9601216</note><identifier type="doi">10.1021/tx9601216</identifier><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>10</number></detail><extent unit="pages"><start>181</start><end>188</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00018" displayLabel="bibbi972327rX00018"><name type="personal"><namePart type="family">NAIR</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">BARBIN</namePart><namePart type="given">A.</namePart></name><name type="personal"><namePart type="family">GUICHARD</namePart><namePart type="given">Y.</namePart></name><name type="personal"><namePart type="family">BARTSCH</namePart><namePart type="given">H.</namePart></name><titleInfo><title>Carcinogenesis</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Nair J. Barbin A. Guichard Y. Bartsch H. Carcinogenesis 1995 16 613 617</note><part><date>1995</date><detail type="volume"><caption>vol.</caption><number>16</number></detail><extent unit="pages"><start>613</start><end>617</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00019" displayLabel="bibbi972327rX00019"><name type="personal"><namePart type="family">AMES</namePart><namePart type="given">B. N.</namePart></name><name type="personal"><namePart type="family">GOLD</namePart><namePart type="given">L. S.</namePart></name><titleInfo><title>Mutat. Res.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Ames B. N. Gold L. S. Mutat. Res. 1991 250 3 16</note><part><date>1991</date><detail type="volume"><caption>vol.</caption><number>250</number></detail><extent unit="pages"><start>3</start><end>16</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00020" displayLabel="bibbi972327rX00020"><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><name type="personal"><namePart type="family">BURCHAM</namePart><namePart type="given">P. C.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Marnett L. J. Burcham P. C. Chem. Res. Toxicol. 1993 6 771 785 10.1021/tx00036a005</note><identifier type="doi">10.1021/tx00036a005</identifier><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>6</number></detail><extent unit="pages"><start>771</start><end>785</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00021" displayLabel="bibbi972327rX00021"><name type="personal"><namePart type="family">LEONARD</namePart><namePart type="given">N. J.</namePart></name><titleInfo><title>Crit. Rev. Biochem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Leonard N. J. Crit. Rev. Biochem. 1984 15 125 199 10.3109/10409238409102299</note><identifier type="doi">10.3109/10409238409102299</identifier><part><date>1984</date><detail type="volume"><caption>vol.</caption><number>15</number></detail><extent unit="pages"><start>125</start><end>199</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00022" displayLabel="bibbi972327rX00022"><name type="personal"><namePart type="family">MüLLER</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">BELAS</namePart><namePart type="given">F. J.</namePart></name><name type="personal"><namePart type="family">BLAIR</namePart><namePart type="given">I. A.</namePart></name><name type="personal"><namePart type="family">GUENGERICH</namePart><namePart type="given">F. P.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Müller M. Belas F. J. Blair I. A. Guengerich F. P. Chem. Res. Toxicol. 1997 10 242 247 10.1021/tx960124i</note><identifier type="doi">10.1021/tx960124i</identifier><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>10</number></detail><extent unit="pages"><start>242</start><end>247</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00023" displayLabel="bibbi972327rX00023"><name type="personal"><namePart type="family">LANGOUëT</namePart><namePart type="given">S.</namePart></name><name type="personal"><namePart type="family">MüLLER</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">GUENGERICH</namePart><namePart type="given">F. P.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Langouët S. Müller M. Guengerich F. P. Biochemistry 1997 36 6069 6079</note><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>36</number></detail><extent unit="pages"><start>6069</start><end>6079</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00024" displayLabel="bibbi972327rX00024"><name type="personal"><namePart type="family">LOWE</namePart><namePart type="given">L. G.</namePart></name><name type="personal"><namePart type="family">GUENGERICH</namePart><namePart type="given">F. P.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Lowe L. G. Guengerich F. P. Biochemistry 1996 35 9840 9849</note><part><date>1996</date><detail type="volume"><caption>vol.</caption><number>35</number></detail><extent unit="pages"><start>9840</start><end>9849</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00025" displayLabel="bibbi972327rX00025"><name type="personal"><namePart type="family">FURGE</namePart><namePart type="given">L. L.</namePart></name><name type="personal"><namePart type="family">GUENGERICH</namePart><namePart type="given">F. P.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Furge L. L. Guengerich F. P. Biochemistry 1997 36 6475 6487</note><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>36</number></detail><extent unit="pages"><start>6475</start><end>6487</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00026" displayLabel="bibbi972327rX00026"><name type="personal"><namePart type="family">MARINELLI</namePart><namePart type="given">E. R.</namePart></name><name type="personal"><namePart type="family">JOHNSON</namePart><namePart type="given">F.</namePart></name><name type="personal"><namePart type="family">IDEN</namePart><namePart type="given">C. R.</namePart></name><name type="personal"><namePart type="family">YU</namePart><namePart type="given">P. L.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Marinelli E. R. Johnson F. Iden C. R. Yu P. L. Chem. Res. Toxicol. 1990 3 49 58 10.1021/tx00013a009</note><identifier type="doi">10.1021/tx00013a009</identifier><part><date>1990</date><detail type="volume"><caption>vol.</caption><number>3</number></detail><extent unit="pages"><start>49</start><end>58</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00027" displayLabel="bibbi972327rX00027"><name type="personal"><namePart type="family">DECORTE</namePart><namePart type="given">B. L.</namePart></name><name type="personal"><namePart type="family">TSAROUHTSIS</namePart><namePart type="given">D.</namePart></name><name type="personal"><namePart type="family">KUCHIMANCHI</namePart><namePart type="given">S.</namePart></name><name type="personal"><namePart type="family">COOPER</namePart><namePart type="given">M. D.</namePart></name><name type="personal"><namePart type="family">HORTON</namePart><namePart type="given">P.</namePart></name><name type="personal"><namePart type="family">HARRIS</namePart><namePart type="given">C. M.</namePart></name><name type="personal"><namePart type="family">HARRIS</namePart><namePart type="given">T. M.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Decorte B. L. Tsarouhtsis D. Kuchimanchi S. Cooper M. D. Horton P. Harris C. M. Harris T. M. Chem. Res. Toxicol. 1996 9 630 637 10.1021/tx9501795</note><identifier type="doi">10.1021/tx9501795</identifier><part><date>1996</date><detail type="volume"><caption>vol.</caption><number>9</number></detail><extent unit="pages"><start>630</start><end>637</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00028" displayLabel="bibbi972327rX00028"><name type="personal"><namePart type="family">BORER</namePart><namePart type="given">P. N.</namePart></name><name type="personal"><namePart type="family">FASMAN</namePart><namePart type="given">G. D.</namePart></name><titleInfo><title>Handbook of Biochemistry and Molecular Biology</title></titleInfo><note type="content-in-line">BorerP. N. (1975) in Handbook of Biochemistry and Molecular Biology (FasmanG. D., Ed.)3rd ed., pp 589−580, CRC Press, Cleveland, OH.</note></relatedItem><relatedItem type="references" ID="bi972327rX00029" displayLabel="bibbi972327rX00029"><name type="personal"><namePart type="family">BENAMIRA</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">SINGH</namePart><namePart type="given">U.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>J. Biol. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Benamira M. Singh U. Marnett L. J. J. Biol. Chem. 1992 267 22392 22400</note><part><date>1992</date><detail type="volume"><caption>vol.</caption><number>267</number></detail><extent unit="pages"><start>22392</start><end>22400</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00030" displayLabel="bibbi972327rX00030"><name type="personal"><namePart type="family">BENAMIRA</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Benamira M. Marnett L. J. Chem. Res. Toxicol. 1993 6 317 327 10.1021/tx00033a011</note><identifier type="doi">10.1021/tx00033a011</identifier><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>6</number></detail><extent unit="pages"><start>317</start><end>327</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00031" displayLabel="bibbi972327rX00031"><name type="personal"><namePart type="family">SAMBROOK</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">FRITSCH</namePart><namePart type="given">E. F.</namePart></name><name type="personal"><namePart type="family">AND MANIATIS</namePart><namePart type="given">T.</namePart></name><titleInfo><title>Molecular Cloning: </title></titleInfo><note type="content-in-line">SambrookJ., FritschE. F., and ManiatisT. (1989) Molecular Cloning: A Laboratory Manual, p A.1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</note></relatedItem><relatedItem type="references" ID="bi972327rX00032" displayLabel="bibbi972327rX00032"><name type="personal"><namePart type="family">SAMBROOK</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">FRITSCH</namePart><namePart type="given">E. F.</namePart></name><name type="personal"><namePart type="family">AND MANIATIS</namePart><namePart type="given">T.</namePart></name><titleInfo><title>Molecular Cloning: </title></titleInfo><note type="content-in-line">SambrookJ., FritschE. F., and ManiatisT. (1989) Molecular Cloning: A Laboratory Manual, p B.15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</note></relatedItem><relatedItem type="references" ID="bi972327rX00033" displayLabel="bibbi972327rX00033"><name type="personal"><namePart type="family">FINK</namePart><namePart type="given">S. P.</namePart></name><name type="personal"><namePart type="family">REDDY</namePart><namePart type="given">G. R.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Fink S. P. Reddy G. R. Marnett L. J. Chem. Res. Toxicol. 1996 9 277 283 10.1021/tx950060w</note><identifier type="doi">10.1021/tx950060w</identifier><part><date>1996</date><detail type="volume"><caption>vol.</caption><number>9</number></detail><extent unit="pages"><start>277</start><end>283</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00034" displayLabel="bibbi972327rX00034"><name type="personal"><namePart type="family">BOOSALIS</namePart><namePart type="given">M. S.</namePart></name><name type="personal"><namePart type="family">PETRUSKA</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">GOODMAN</namePart><namePart type="given">M. F.</namePart></name><titleInfo><title>J. Biol. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Boosalis M. S. Petruska J. Goodman M. F. J. Biol. Chem. 1987 262 14689 14696</note><part><date>1987</date><detail type="volume"><caption>vol.</caption><number>262</number></detail><extent unit="pages"><start>14689</start><end>14696</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00035" displayLabel="bibbi972327rX00035"><name type="personal"><namePart type="family">MAXAM</namePart><namePart type="given">A. M.</namePart></name><name type="personal"><namePart type="family">GILBERT</namePart><namePart type="given">W.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Maxam A. M. Gilbert W. Proc. Natl. Acad. Sci. U.S.A. 1977 74 560 564 10.1073/pnas.74.2.560</note><identifier type="doi">10.1073/pnas.74.2.560</identifier><part><date>1977</date><detail type="volume"><caption>vol.</caption><number>74</number></detail><extent unit="pages"><start>560</start><end>564</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00036" displayLabel="bibbi972327rX00036"><name type="personal"><namePart type="family">MAXAM</namePart><namePart type="given">A. M.</namePart></name><name type="personal"><namePart type="family">GILBERT</namePart><namePart type="given">W.</namePart></name><titleInfo><title>Methods Enzymol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Maxam A. M. Gilbert W. Methods Enzymol. 1980 65 499 560</note><part><date>1980</date><detail type="volume"><caption>vol.</caption><number>65</number></detail><extent unit="pages"><start>499</start><end>560</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00037" displayLabel="bibbi972327rX00037"><name type="personal"><namePart type="family">FRIEDMAN</namePart><namePart type="given">T.</namePart></name><name type="personal"><namePart type="family">BROWN</namePart><namePart type="given">D. H.</namePart></name><titleInfo><title>Nucleic Acids Res.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Friedman T. Brown D. H. Nucleic Acids Res. 1978 5 615 622</note><part><date>1978</date><detail type="volume"><caption>vol.</caption><number>5</number></detail><extent unit="pages"><start>615</start><end>622</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00038" displayLabel="bibbi972327rX00038"><name type="personal"><namePart type="family">SATTSANGI</namePart><namePart type="given">P. D.</namePart></name><name type="personal"><namePart type="family">LEONARD</namePart><namePart type="given">N. J.</namePart></name><name type="personal"><namePart type="family">FRIHART</namePart><namePart type="given">C. R.</namePart></name><titleInfo><title>J. Org. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Sattsangi P. D. Leonard N. J. Frihart C. R. J. Org. Chem. 1977 42 3292 3296 10.1021/jo00440a020</note><identifier type="doi">10.1021/jo00440a020</identifier><part><date>1977</date><detail type="volume"><caption>vol.</caption><number>42</number></detail><extent unit="pages"><start>3292</start><end>3296</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00039" displayLabel="bibbi972327rX00039"><name type="personal"><namePart type="family">JOHNSON</namePart><namePart type="given">K. A.</namePart></name><name type="personal"><namePart type="family">FINK</namePart><namePart type="given">S. P.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>J. Biol. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Johnson K. A. Fink S. P. Marnett L. J. J. Biol. Chem. 1997 272 11434 11438 10.1074/jbc.272.17.11434</note><identifier type="doi">10.1074/jbc.272.17.11434</identifier><part><date>1997</date><detail type="volume"><caption>vol.</caption><number>272</number></detail><extent unit="pages"><start>11434</start><end>11438</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00040" displayLabel="bibbi972327rX00040"><name type="personal"><namePart type="family">HASHIM</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">MARNETT</namePart><namePart type="given">L. J.</namePart></name><titleInfo><title>J. Biol. Chem.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Hashim M. Marnett L. J. J. Biol. Chem. 1996 271 9160 9165 10.1074/jbc.271.15.9160</note><identifier type="doi">10.1074/jbc.271.15.9160</identifier><part><date>1996</date><detail type="volume"><caption>vol.</caption><number>271</number></detail><extent unit="pages"><start>9160</start><end>9165</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00041" displayLabel="bibbi972327rX00041"><name type="personal"><namePart type="family">DOSANJH</namePart><namePart type="given">M. K.</namePart></name><name type="personal"><namePart type="family">CHENNA</namePart><namePart type="given">A.</namePart></name><name type="personal"><namePart type="family">KIM</namePart><namePart type="given">E.</namePart></name><name type="personal"><namePart type="family">FRAENKEL-CONRAT</namePart><namePart type="given">H.</namePart></name><name type="personal"><namePart type="family">SAMSON</namePart><namePart type="given">L.</namePart></name><name type="personal"><namePart type="family">SINGER</namePart><namePart type="given">B.</namePart></name><titleInfo><title>Proc. Natl. Acad. Sci. U.S.A.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Dosanjh M. K. Chenna A. Kim E. Fraenkel-Conrat H. Samson L. Singer B. Proc. Natl. Acad. Sci. U.S.A. 1994 91 1024 1028 10.1073/pnas.91.3.1024</note><identifier type="doi">10.1073/pnas.91.3.1024</identifier><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>91</number></detail><extent unit="pages"><start>1024</start><end>1028</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00042" displayLabel="bibbi972327rX00042"><name type="personal"><namePart type="family">SHAPIRO</namePart><namePart type="given">R.</namePart></name><name type="personal"><namePart type="family">COHEN</namePart><namePart type="given">B. I.</namePart></name><name type="personal"><namePart type="family">SHIUEY</namePart><namePart type="given">S. J.</namePart></name><name type="personal"><namePart type="family">MAURER</namePart><namePart type="given">H.</namePart></name><titleInfo><title>Biochemistry</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Shapiro R. Cohen B. I. Shiuey S. J. Maurer H. Biochemistry 1969 8 238 245</note><part><date>1969</date><detail type="volume"><caption>vol.</caption><number>8</number></detail><extent unit="pages"><start>238</start><end>245</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00043" displayLabel="bibbi972327rX00043"><name type="personal"><namePart type="family">FOILES</namePart><namePart type="given">P. G.</namePart></name><name type="personal"><namePart type="family">AKERKAR</namePart><namePart type="given">S. A.</namePart></name><name type="personal"><namePart type="family">MIGLIETTA</namePart><namePart type="given">L. M.</namePart></name><name type="personal"><namePart type="family">CHUNG</namePart><namePart type="given">F. L.</namePart></name><titleInfo><title>Carcinogenesis</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Foiles P. G. Akerkar S. A. Miglietta L. M. Chung F. L. Carcinogenesis 1990 11 2059 2061</note><part><date>1990</date><detail type="volume"><caption>vol.</caption><number>11</number></detail><extent unit="pages"><start>2059</start><end>2061</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00044" displayLabel="bibbi972327rX00044"><name type="personal"><namePart type="family">CHUNG</namePart><namePart type="given">F. L.</namePart></name><name type="personal"><namePart type="family">KRZEMINSKI</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">WANG</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">CHEN</namePart><namePart type="given">H. J. C.</namePart></name><name type="personal"><namePart type="family">PROKOPCZYK</namePart><namePart type="given">B.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Chung F. L. Krzeminski J. Wang M. Chen H. J. C. Prokopczyk B. Chem. Res. Toxicol. 1994 7 62 67 10.1021/tx00037a009</note><identifier type="doi">10.1021/tx00037a009</identifier><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>7</number></detail><extent unit="pages"><start>62</start><end>67</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00045" displayLabel="bibbi972327rX00045"><name type="personal"><namePart type="family">CHUNG</namePart><namePart type="given">F.-L.</namePart></name><name type="personal"><namePart type="family">HECHT</namePart><namePart type="given">S. S.</namePart></name><titleInfo><title>Cancer Res.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Chung F.-L. Hecht S. S. Cancer Res. 1983 43 1230 1235</note><part><date>1983</date><detail type="volume"><caption>vol.</caption><number>43</number></detail><extent unit="pages"><start>1230</start><end>1235</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00046" displayLabel="bibbi972327rX00046"><name type="personal"><namePart type="family">CHUNG</namePart><namePart type="given">F. L.</namePart></name><name type="personal"><namePart type="family">CHEN</namePart><namePart type="given">H. J. C.</namePart></name><name type="personal"><namePart type="family">GUTTENPLAN</namePart><namePart type="given">J. B.</namePart></name><name type="personal"><namePart type="family">NISHIKAWA</namePart><namePart type="given">A.</namePart></name><name type="personal"><namePart type="family">HARD</namePart><namePart type="given">G. C.</namePart></name><titleInfo><title>Carcinogenesis</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">Chung F. L. Chen H. J. C. Guttenplan J. B. Nishikawa A. Hard G. C. Carcinogenesis 1993 14 2073 2077</note><part><date>1993</date><detail type="volume"><caption>vol.</caption><number>14</number></detail><extent unit="pages"><start>2073</start><end>2077</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rX00047" displayLabel="bibbi972327rX00047"><name type="personal"><namePart type="family">EL GHISSASSI</namePart><namePart type="given">F.</namePart></name><name type="personal"><namePart type="family">BARBIN</namePart><namePart type="given">A.</namePart></name><name type="personal"><namePart type="family">NAIR</namePart><namePart type="given">J.</namePart></name><name type="personal"><namePart type="family">BARTSCH</namePart><namePart type="given">H.</namePart></name><titleInfo><title>Chem. Res. Toxicol.</title></titleInfo><genre type="journal">journal</genre><note type="content-in-line">El Ghissassi F. Barbin A. Nair J. Bartsch H. Chem. Res. Toxicol. 1995 8 278 283 10.1021/tx00044a013</note><identifier type="doi">10.1021/tx00044a013</identifier><part><date>1995</date><detail type="volume"><caption>vol.</caption><number>8</number></detail><extent unit="pages"><start>278</start><end>283</end></extent></part></relatedItem><relatedItem type="references" ID="bi972327rb00001" displayLabel="bibbi972327rb00001"><titleInfo><title>Abbreviations: ε, etheno; Gua or G, guanine; 1,N2-ε-Gua, 1,N2-ethenoguanine (or 9-oxoimidazo[1,2-a]purine); ethanoGua, 1,N2-ethanoguanine (5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine); HO-ethanoGua, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazopurine; dGuo, deoxyguanosine; ethanodGuo, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine 2-deoxyribosyl nucleoside; M1Gua, pyrimido[1,2-a]purin-10(3H)one; propanoGua, 1,N2-propanoguanine; propanodGuo, 1,N2-propano-2‘-deoxyguanosine; HO-propanoGua, 8-hydroxypropanoGua; CGE, capillary gel electrophoresis; MOPS, 3-(N-morphilino)propanesulfonic acid; SSC, saline sodium citrate buffer (15 mM sodium citrate buffer (pH 7.0) containing 150 mM NaCl); SSPE, 10 sodium phosphate buffer (pH 7.4) containing 1.0 mM EDTA and 150 mM NaCl; Kf-,E. colipolymerase I (Klenow fragment) exo-; pol II-,E. colipolymerase II exo-; T7-, bacteriophage T7 polymerase exo-/thioredoxin complex; HIV RT, human immunodeficiency virus-1 reverse transcriptase; ES MS, electrospray mass spectrometry; Tris, tris(hydroxymethyl)aminomethane; EDTA, (ethylenedinitrilo)acetic acid.</title></titleInfo><note type="content-in-line">Abbreviations: ε, etheno; Gua or G, guanine; 1,N2-ε-Gua, 1,N2-ethenoguanine (or 9-oxoimidazo[1,2-a]purine); ethanoGua, 1,N2-ethanoguanine (5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine); HO-ethanoGua, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazopurine; dGuo, deoxyguanosine; ethanodGuo, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine 2-deoxyribosyl nucleoside; M1Gua, pyrimido[1,2-a]purin-10(3H)one; propanoGua, 1,N2-propanoguanine; propanodGuo, 1,N2-propano-2‘-deoxyguanosine; HO-propanoGua, 8-hydroxypropanoGua; CGE, capillary gel electrophoresis; MOPS, 3-(N-morphilino)propanesulfonic acid; SSC, saline sodium citrate buffer (15 mM sodium citrate buffer (pH 7.0) containing 150 mM NaCl); SSPE, 10 sodium phosphate buffer (pH 7.4) containing 1.0 mM EDTA and 150 mM NaCl; Kf-, E. coli polymerase I (Klenow fragment) exo-; pol II-, E. coli polymerase II exo-; T7-, bacteriophage T7 polymerase exo-/thioredoxin complex; HIV RT, human immunodeficiency virus-1 reverse transcriptase; ES MS, electrospray mass spectrometry; Tris, tris(hydroxymethyl)aminomethane; EDTA, (ethylenedinitrilo)acetic acid.</note></relatedItem><relatedItem type="references" ID="bi972327rb00002" displayLabel="bibbi972327rb00002"><titleInfo><title>Some degradation of the primer was observed with HIV RT, when the template contained either 1,N2-ε-Gua or ethanoGua (FigureD). The basis of this is unknown, but similar results have been seen with other blocking lesions [23; M.-S. Kim and F. P. Guengerich (Chem. Res. Toxicol., in press)] and with several commercial preparations of HIV RT (with M1Gua).</title></titleInfo><note type="content-in-line">Some degradation of the primer was observed with HIV RT, when the template contained either 1,N2-ε-Gua or ethanoGua (Figure D). The basis of this is unknown, but similar results have been seen with other blocking lesions [23; M.-S. Kim and F. P. Guengerich (Chem. Res. Toxicol., in press)] and with several commercial preparations of HIV RT (with M1Gua).</note></relatedItem><relatedItem type="references" ID="bi972327rb00003" displayLabel="bibbi972327rb00003"><titleInfo><title>M. S. Kim and F. P. Guengerich (Chem. Res. Toxicol., in press).</title></titleInfo><note type="content-in-line">M. S. Kim and F. P. Guengerich (Chem. Res. Toxicol., in press).</note></relatedItem><identifier type="istex">004E2BD6D48F49FB7F81EBCFA16DC6B6631F8A9F</identifier><identifier type="ark">ark:/67375/TPS-P1V0PCZ4-2</identifier><identifier type="DOI">10.1021/bi972327r</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-P1V0PCZ4-2/record.json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-P1V0PCZ4-2/annexes.gif</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/004E2BD6D48F49FB7F81EBCFA16DC6B6631F8A9F/annexes/tiff</uri></json:item><json:item><extension>eps</extension><original>true</original><mimetype>image/x-eps</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-P1V0PCZ4-2/annexes.eps</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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|texte= Misincorporation of Nucleotides opposite Five-Membered Exocyclic Ring Guanine Derivatives by Escherichia coli Polymerases in Vitro and in Vivo: 1,N2-Ethenoguanine, 5,6,7,9-Tetrahydro-9-oxoimidazo[1,2-a]purine, and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine†
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