Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†
Identifieur interne : 000615 ( Istex/Corpus ); précédent : 000614; suivant : 000616Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†
Auteurs : Sophie Langouët ; Michael Müller ; F. Peter GuengerichSource :
- Biochemistry [ 0006-2960 ] ; 1997.
Abstract
1,N2-Ethenoguanine (1,N2-ε-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5‘-CAGTGGGTG*TCCGAATTGA-3‘, were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H218O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3‘-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, −1 and −2 base frame shifts were detected with both 1,N2-ε-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in kcat for all dNTP incorporations compared to the normal G·dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.
Url:
DOI: 10.1021/bi962526v
Links to Exploration step
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†</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></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: Abt. Arbeits-und Sozialmedizinder Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</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. 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:2010D51B69180BED3FDB60A600FBAF86BADAEA41</idno><date when="1997" year="1997">1997</date><idno type="doi">10.1021/bi962526v</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-69F58Q91-C/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000615</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000615</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Misincorporation of dNTPs Opposite 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine in Oligonucleotides by
<hi rend="italic">Escherichia coli</hi> Polymerases I exo<hi rend="superscript">-</hi> and II exo<hi rend="superscript">-</hi>, T7 Polymerase exo<hi rend="superscript">-</hi>, Human
Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β<ref type="bib" target="#bi962526vAF2"><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></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: Abt. Arbeits-und Sozialmedizinder Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</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. 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">1997</date><date type="published">1997</date><biblScope unit="vol">36</biblScope><biblScope unit="issue">20</biblScope><biblScope unit="page" from="6069">6069</biblScope><biblScope unit="page" to="6079">6079</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">1,N2-Ethenoguanine (1,N2-ε-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5‘-CAGTGGGTG*TCCGAATTGA-3‘, were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H218O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3‘-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, −1 and −2 base frame shifts were detected with both 1,N2-ε-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in kcat for all dNTP incorporations compared to the normal G·dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>polymerase</json:string><json:string>adduct</json:string><json:string>guengerich</json:string><json:string>primer</json:string><json:string>misincorporation</json:string><json:string>biochemistry</json:string><json:string>hplc</json:string><json:string>chem</json:string><json:string>dntps</json:string><json:string>nucleoside</json:string><json:string>oligonucleotides</json:string><json:string>diastereomers</json:string><json:string>oligonucleotide</json:string><json:string>oligomers</json:string><json:string>mmol</json:string><json:string>oligomer</json:string><json:string>frameshift</json:string><json:string>dctp</json:string><json:string>marnett</json:string><json:string>dthd</json:string><json:string>toxicol</json:string><json:string>dguo</json:string><json:string>dntp</json:string><json:string>dado</json:string><json:string>biol</json:string><json:string>propanog</json:string><json:string>base frameshift</json:string><json:string>dcyd</json:string><json:string>hashim</json:string><json:string>miscoding</json:string><json:string>datp</json:string><json:string>etheno</json:string><json:string>deoxyribose</json:string><json:string>assay</json:string><json:string>lowe guengerich</json:string><json:string>derivative</json:string><json:string>hplc analysis</json:string><json:string>misincorporation frequency</json:string><json:string>nucleotide sequence analysis</json:string><json:string>room temperature</json:string><json:string>incorporation</json:string><json:string>unmodified</json:string><json:string>insertion</json:string><json:string>dctp opposite</json:string><json:string>flow rate</json:string><json:string>incorporation opposite</json:string><json:string>adduct site</json:string><json:string>etheno adduct biochemistry</json:string><json:string>solvent system</json:string><json:string>vanderbilt university school</json:string><json:string>mass spectrum</json:string><json:string>primer extension</json:string><json:string>several polymerase</json:string><json:string>exocyclic ring</json:string><json:string>polymerase extension assay</json:string><json:string>nucleic acid</json:string><json:string>bacterial study</json:string><json:string>oligonucleotide synthesis</json:string><json:string>template</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></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: Abt. Arbeits-und Sozialmedizinder Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</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. 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-69F58Q91-C</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>1,N2-Ethenoguanine (1,N2-ε-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5‘-CAGTGGGTG*TCCGAATTGA-3‘, were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H218O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3‘-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, −1 and −2 base frame shifts were detected with both 1,N2-ε-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in kcat for all dNTP incorporations compared to the normal G·dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.</abstract><qualityIndicators><score>10</score><pdfWordCount>7635</pdfWordCount><pdfCharCount>47138</pdfCharCount><pdfVersion>1.1</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>694</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>261</abstractWordCount><abstractCharCount>1936</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†</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>36</volume><issue>20</issue><pages><first>6069</first><last>6079</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-69F58Q91-C</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><json:string>4 - agronomie. sciences du sol et productions vegetales</json:string></inist></categories><publicationDate>1997</publicationDate><copyrightDate>1997</copyrightDate><doi><json:string>10.1021/bi962526v</json:string></doi><id>2010D51B69180BED3FDB60A600FBAF86BADAEA41</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-69F58Q91-C/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-69F58Q91-C/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-69F58Q91-C/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-69F58Q91-C/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Misincorporation of dNTPs Opposite 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine in Oligonucleotides by
<hi rend="italic">Escherichia coli</hi> Polymerases I exo<hi rend="superscript">-</hi> and II exo<hi rend="superscript">-</hi>, T7 Polymerase exo<hi rend="superscript">-</hi>, Human
Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β<ref type="bib" target="#bi962526vAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 1997 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">1997</date><date type="Copyright" when="1997">1997</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 dNTPs Opposite 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-Ethenoguanine and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine in Oligonucleotides by
<hi rend="italic">Escherichia coli</hi> Polymerases I exo<hi rend="superscript">-</hi> and II exo<hi rend="superscript">-</hi>, T7 Polymerase exo<hi rend="superscript">-</hi>, Human
Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β<ref type="bib" target="#bi962526vAF2"><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></author><author xml:id="author-0001"><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="bi962526vAF3"><ref>‡</ref><p>
Current address: Abt. Arbeits-und Sozialmedizin
der Georg-August-Universität Göttingen, Waldweg 37, D-37073
Göttingen,
Germany.</p></note></author><author xml:id="author-0002" 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. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@toxicology.mc. vanderbilt.edu.</affiliation></author><idno type="istex">2010D51B69180BED3FDB60A600FBAF86BADAEA41</idno><idno type="ark">ark:/67375/TPS-69F58Q91-C</idno><idno type="DOI">10.1021/bi962526v</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">1997</date><date type="published">1997</date><biblScope unit="vol">36</biblScope><biblScope unit="issue">20</biblScope><biblScope unit="page" from="6069">6069</biblScope><biblScope unit="page" to="6079">6079</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>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) and
5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine
(HO-ethanoGua) are two modified bases formed in the reaction of DNA
with 2-chlorooxirane, the epoxide
derivative of vinyl chloride. The oligonucleotides (19-mers),
5‘-CAGTGGGTG*TCCGAATTGA-3‘, were
prepared, with each of these modified bases substituted for G at G*.
HO-ethanodeoxyguanosine exists
predominantly as a mixture of diastereomers of the closed cyclic
hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<hi rend="italic">a</hi>]purine, shown by
H<hi rend="subscript">2</hi><hi rend="superscript">18</hi>O experiments to be in equilibrium with the
open
form, <hi rend="italic">N</hi><hi rend="superscript">2</hi>-(2-oxoethyl)Gua. Both adducts
retarded the 3‘-extension of a complementary 10-mer primer
by
all of the polymerases examined, but in every case, some full-length
product was obtained. Nucleotide
sequence analysis indicated misincorporation of dGTP and dATP across
from both 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases.
Similar results were obtained
when the abilities of the polymerases to incorporate a single dNTP were
evaluated. In addition, −1 and
−2 base frame shifts were detected with both
1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua and HO-ethanoGua with some of the
polymerases.
Steady-state kinetic experiments with <hi rend="italic">Escherichia coli</hi>
polymerase I exo<hi rend="superscript">-</hi> and T7 polymerase
exo<hi rend="superscript">-</hi>/thioredoxin showed large decreases in <hi rend="italic">k</hi><hi rend="subscript">cat</hi> for
all dNTP incorporations compared to the normal G·dCTP
pair and high misincorporation frequencies for dATP and dGTP with both
adducts (compared to dCTP).
Collectively, the results indicate that both of these adducts have
considerable miscoding potential with
some of these polymerases, that there are a number of differences
between the 1,<hi rend="italic">N</hi><hi rend="superscript">2</hi>-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the
elements of water), and that
misincorporation of dNTPs at a single modified base can vary
considerably among different polymerases
even in the absence of exonuclease activity.
</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/bi962526v</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 dNTPs Opposite 1,<italic toggle="yes">N</italic><sup>2</sup>-Ethenoguanine and
5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine in Oligonucleotides by
<italic toggle="yes">Escherichia coli</italic> Polymerases I exo<sup>-</sup> and II exo<sup>-</sup>, T7 Polymerase exo<sup>-</sup>, Human
Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β<xref rid="bi962526vAF2"><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></contrib><contrib contrib-type="author"><name name-style="western"><surname>Müller</surname><given-names>Michael</given-names></name><xref rid="bi962526vAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Guengerich</surname><given-names>F. Peter</given-names></name><xref rid="bi962526vAF1">*</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="bi962526vAF3"><label>‡</label><p>
Current address: Abt. Arbeits-und Sozialmedizin
der Georg-August-Universität Göttingen, Waldweg 37, D-37073
Göttingen,
Germany.</p></fn><corresp id="bi962526vAF1">
Address correspondence to this author. Telephone: (615)
322-2261. Fax: (615) 322-3141. E-mail:
guengerich@toxicology.mc.
vanderbilt.edu.</corresp></author-notes><pub-date pub-type="epub"><day>20</day><month>05</month><year>1997</year></pub-date><pub-date pub-type="ppub"><day>20</day><month>05</month><year>1997</year></pub-date><volume>36</volume><issue>20</issue><fpage>6069</fpage><lpage>6079</lpage><supplementary-material xlink:href="bi6069.pdf" orientation="portrait" position="float"></supplementary-material><history><date date-type="received"><day>08</day><month>10</month><year>1996</year></date><date date-type="rev-recd"><day>17</day><month>03</month><year>1997</year></date><date date-type="issue-pub"><day>20</day><month>05</month><year>1997</year></date></history><permissions><copyright-statement>Copyright © 1997 American Chemical Society</copyright-statement><copyright-year>1997</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>1,<italic toggle="yes">N</italic><sup>2</sup>-Ethenoguanine
(1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua) and
5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine
(HO-ethanoGua) are two modified bases formed in the reaction of DNA
with 2-chlorooxirane, the epoxide
derivative of vinyl chloride. The oligonucleotides (19-mers),
5‘-CAGTGGGTG*TCCGAATTGA-3‘, were
prepared, with each of these modified bases substituted for G at G*.
HO-ethanodeoxyguanosine exists
predominantly as a mixture of diastereomers of the closed cyclic
hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-<italic toggle="yes">a</italic>]purine, shown by
H<sub>2</sub><sup>18</sup>O experiments to be in equilibrium with the
open
form, <italic toggle="yes">N</italic><sup>2</sup>-(2-oxoethyl)Gua. Both adducts
retarded the 3‘-extension of a complementary 10-mer primer
by
all of the polymerases examined, but in every case, some full-length
product was obtained. Nucleotide
sequence analysis indicated misincorporation of dGTP and dATP across
from both 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases.
Similar results were obtained
when the abilities of the polymerases to incorporate a single dNTP were
evaluated. In addition, −1 and
−2 base frame shifts were detected with both
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua with some of the
polymerases.
Steady-state kinetic experiments with <italic toggle="yes">Escherichia coli</italic>
polymerase I exo<sup>-</sup> and T7 polymerase
exo<sup>-</sup>/thioredoxin showed large decreases in <italic toggle="yes">k</italic><sub>cat</sub> for
all dNTP incorporations compared to the normal G·dCTP
pair and high misincorporation frequencies for dATP and dGTP with both
adducts (compared to dCTP).
Collectively, the results indicate that both of these adducts have
considerable miscoding potential with
some of these polymerases, that there are a number of differences
between the 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the
elements of water), and that
misincorporation of dNTPs at a single modified base can vary
considerably among different polymerases
even in the absence of exonuclease activity.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi962526v</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi962526vAF2"><label>†</label><p>
This research was supported in part by United
States Public Health
Service Grants R35 CA44353 and P30 ES00267. M.M. was
supported
in part by a fellowship from the Deutsche
Forschungsgemeinschaft.</p></notes><notes id="bi962526vAF7"><label>✗</label><p>
Abstract published in <italic toggle="yes">Advance ACS
Abstracts,</italic> May 1, 1997.</p></notes></front><body><sec id="d7e209"><title></title><p>The etheno (ε)<xref rid="atyp_ref1" ref-type="bibr"></xref> derivatives of purines and
pyrimidines
have an extra five-membered, unsaturated ring containing
two added carbons (Scheme <xref rid="bi962526vh00001"></xref>) (Singer & Bartsch,
1986).
These ε compounds were originally discovered as
modified
tRNA bases in tRNAs (Nakanishi et al., 1970) and as
products of RNA treated with 2-haloacetaldehydes (Kochetkov et al., 1971; Barrio et al., 1972). For instance,
6-methylimidazo[1,2-<italic toggle="yes">a</italic>]purine (6-methylwyosine or
6-methyl-1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua) is a naturally occurring tRNA
base (Agris,
1996). The strong fluorescence of ε-Ade has been
utilized
in studies of the interaction of NADP<sup>+</sup>, ATP, and
other
derivatives with proteins (Leonard, 1984). The
hepatocarcinogen vinyl chloride is oxidized to 2-chlorooxirane,
which
reacts with DNA to form 1,<italic toggle="yes">N</italic><sup>6</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, 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, and HO-ethanoGua (Guengerich
et al.,
1993, 1994; Müller et al., 1997). Other carcinogens
[e.g.,
acrylonitrile and urethan (Guengerich et al., 1981a;
Guengerich & Kim, 1991)] can be oxidized to
similar epoxides that
react with DNA to form these same ε products. Of
particular
interest is the recent discovery that low but finite levels
of
1,<italic toggle="yes">N</italic><sup>6</sup>-ε-Ade and
3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyt have been found in DNA
prepared
from experimental animals and humans that were not exposed
to any of the known precursors (Fedtke et al., 1990).
The
origin of these may be products of lipid peroxidation (El
Ghissassi et al., 1995) or possibly halogenated drinking
water
contaminants (Kronberg et al., 1992).
<fig id="bi962526vh00001" position="float" fig-type="scheme" orientation="portrait"><label>1</label><caption><p>Structures of Five-Membered Ring Exocyclic Gua DNA Adducts</p></caption><graphic xlink:href="bi962526vh00001.gif" position="float" orientation="portrait"></graphic></fig></p><p>Concern arises about the biological significance of the
ε
bases because of their presence in DNA and the carcinogenicity of chemicals that give rise to these. Because
the
reaction of DNA with appropriately functionalized two-carbon compounds yields all of these ε adducts, studies
with
individual bases incorporated in oligonucleotides and
vectors
are necessary to evaluate the ability of each base to
cause
misincorporation. 1,<italic toggle="yes">N</italic><sup>6</sup>-ε-Ade has been
found to be weakly
mutagenic in bacteria (Basu et al., 1993).
3,<italic toggle="yes">N</italic><sup>4</sup>-ε-Cyt was
reported to be highly mutagenic in one bacterial study
(Palejwala et al., 1993) but weakly mutagenic in others
(Basu
et al., 1993; Moriya et al., 1994). However, in the
latter
study, it was found to be considerably more mutagenic in a
mammalian cell system (Moriya et al., 1994).
<italic toggle="yes">N</italic><sup>2</sup>,3-ε-Gua
appears to be rather mutagenic as judged by the results of
two bacterial studies in which <italic toggle="yes">N</italic><sup>2</sup>,3-ε-dGTP was
incorporated
into plasmids (Cheng et al., 1991; Singer et al., 1987);
however, the instability of the glycosidic bond (Khazanchi
et al., 1993; Kusmierek et al., 1989) has precluded more
systematic studies of the characterization of the
miscoding
properties of this base.
</p><p>In the course of our studies on the reaction of 2-halooxiranes with DNA (Guengerich et al., 1979, 1981b;
Guengerich & Raney, 1992), we characterized HO-ethanoGua as a major product of the reaction of 2-chlorooxirane
with Gua derivatives (Guengerich et al., 1993).
Recently,
we have found that treatment of DNA with 2-chlorooxirane
yields products in the concentration order
<italic toggle="yes">N</italic><sup>7</sup>-(2-oxoethyl)Gua
≫ 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 (Müller et al., 1997).
We were interested
in the miscoding potential of HO-ethanoGua and comparison
to 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, which differs only in the
absence of the
elements of H<sub>2</sub>O and, to our knowledge, has not
been
examined for miscoding properties. We report the
synthesis
of oligomers containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua at
a defined position and misincorporation studies with five
model polymerases. Some comparisons are made to
studies
done with the homologs containing six-membered rings,
which are also found in DNA both before and after
treatment
of animals with carcinogens (Chaudhary et al., 1994; Nath
& Chung, 1994).
</p></sec><sec id="d7e334"><title>Experimental Procedures</title><sec id="d7e337"><title>Chemicals
</title><p>Most chemicals were purchased from Aldrich Chemical
Co. (Milwaukee, WI). 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo was a
gift of L. J.
Marnett (Department of Biochemistry, Vanderbilt University
School of Medicine). H<sub>2</sub><sup>18</sup>O was supplied by
Cambridge
Isotope Laboratories (Cambridge, MA). Reagents for
oligonucleotide synthesis were purchased from PerSeptive
Biosystems (Framingham, MA).
</p></sec></sec><sec id="d7e352"><title></title><sec id="d7e354"><title>Instrumental and Chromatographic Analysis
</title><p><italic toggle="yes">TLC.</italic> TLC was done with silica gel F 254
(Merck,
Gibbstown, NJ) as the adsorbent on glass plates. The
separated compounds were visualized under UV light (254
nm) or by staining with an anisaldehyde/H<sub>2</sub>SO<sub>4</sub>
mixture and
subsequent heating. Column chromatography was
performed
with silica gel 60 (70−230 mesh, Merck).
</p><p><italic toggle="yes">HPLC.</italic> HPLC was done with reversed-phase
octadecylsilane columns: 10 × 250 mm Beckman Ultrasphere, 5 μm
(Beckman, San Ramon, CA) for analysis and preparative
isolation of base and nucleoside adducts; 4.6 × 250 mm
and
10 × 250 mm YMC-Pack ODS-AQ, 5 μm (YMC, Wilmington, NC) for DNA oligomer purification and enzymatic
digest analysis. The columns were connected to a
Spectra-Physics 8700 pumping system (Thermo-Separation Products,
Piscataway, NJ), with the effluent passing through a
Hewlett-Packard 1040A diode array detector (Hewlett-Packard, Palo
Alto, CA). The separation of the base and nucleoside
adducts involved increasing gradients of CH<sub>3</sub>OH
(solvent
B) in 50 mM NH<sub>4</sub>HCO<sub>2</sub> at pH 5.0 (solvent A),
usually
increasing from 0 to 50% CH<sub>3</sub>OH over 25 min, with a
flow
rate of 2.5 mL min<sup>-1</sup>. Initial purification of the
oligonucleotides with this solvent system was achieved with the
following gradient: 0 min (99% A, 1% B), 50 min (70%
A,
30% B), 55 min (10% A, 90% B), and 60 min (99% A, 1%
B) at a flow rate of 2.5 mL min<sup>-1</sup>. To increase
purity to
>99%, a second HPLC step was performed with the
following gradient: 0 min (75% A, 25% B) and 50 min
(70%
A, 30% B), as well as a 20% (w/v) polyacrylamide gel
electrophoresis purification. Enzymatic digests of the
oligonucleotides (<italic toggle="yes">vide infra</italic>) were analyzed on the
analytical
YMC-Pack column with the above solvent system at a flow
rate of 1.0 mL min<sup>-1</sup> and the following gradients:
unmodified and 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua-containing 19-mers, 0 min
(99% A, 1%
B), 30 min (80% A, 20% B), 45 min (50% A, 50% B), and
50 min (99% A, 1% B); 10-mer primer and HO-ethanoGua-containing 19-mer, 0 min (99% A, 1% B), 40 min (80% A,
20% B), 55 min (50% A, 50% B), and 60 min (99% A, 1%
B).
</p><p><italic toggle="yes">CGE.</italic> Oligonucleotide purity was evaluated using
a
Beckman P/ACE 2000 instrument (Beckman, Fullerton, CA)
using the “ssDNA 100” gel capillary and
“TRIS-borate-urea
buffer” from the manufacturer. Samples were applied at
−5
kV and run at −10 kV (30 °C).
</p><p><italic toggle="yes">UV and CD Spectroscopy.</italic> UV spectra were
recorded
using a a modified Cary 14/OLIS instrument (On-Line
Instrument Systems, Bogart, GA). CD spectra were
recorded
using a JASCO J-720 spectropolarimeter (Japan Applied
Spectroscopic Co., Tokyo, Japan).
</p><p><italic toggle="yes">NMR.</italic> <sup>1</sup>H-NMR spectra were recorded in
H<sub>2</sub>O/<sup>2</sup>H<sub>2</sub>O
mixtures or (C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO using a
Bruker AM 400 spectrometer
(Bruker, Billerica, MA) in the Vanderbilt facility.
<sup>31</sup>P-NMR
spectra were acquired in C<sup>2</sup>H<sub>3</sub>CN on a Bruker AC
300
instrument with 85% H<sub>3</sub>PO<sub>4</sub> as the external
standard.
</p><p><italic toggle="yes">MS.</italic> Mass spectra of the base and nucleoside
DNA
adducts were collected on a Kratos Concept II HH
instrument
(Kratos, Manchester, U.K.) with FAB ionization and a
mixture of glycerol, (CH<sub>3</sub>)<sub>2</sub>SO, and
3-nitrobenzyl alcohol as
the matrix. Oligonucleotide ES mass spectra were
obtained
on a Finnigan TSQ 7000 ES instrument (Finnigan, San Jose,
CA) equipped with a Unix computer system and deconvolution software.
</p><p>HPLC/ES MS studies were conducted using the Finnigan
TSQ 7000 triple-quadrupole mass spectrometer operating in
the positive ion mode with an electrospray needle voltage
of 4.5 kV. N<sub>2</sub> was used as the sheath gas (60 psi) to
assist
with nebulization and as the auxiliary gas (15 psi) to
assist
with desolvation. The stainless steel capillary was
heated
to 220 and 200 °C, respectively, to provide optimal
desolvation, and the ESI interface and mass spectrometer parameters were optimized to obtain maximum sensitivity
(Müller
et al., 1997). The tube lens and the heated capillary
were
operated at 74.5 and 20.0 V, respectively, and the
electron
multiplier was set at 1700 V. Selected reaction
monitoring
experiments were conducted by monitoring <italic toggle="yes">m/z</italic> for
each
protonated molecular ion. For the analysis of
nucleosides,
the oligomer was digested according to the method of
Chaudhary et al. (1994). The digest was filtered through
a
0.22 μm filter, and 250 μL aliquots were analyzed by
HPLC/MS. HPLC involved the use of a Hewlett-Packard 1090
HPLC pumping system, connected to a Phenomenex Partisil
ODS-3 reversed-phase column (3.2 × 250 mm, 5 μm,
Phenomenex, Torrance, CA). The solvent system
consisted
of 10 mM NH<sub>4</sub>CH<sub>3</sub>CO<sub>2</sub> buffer at pH 5.5
(A) and 0.05%
CH<sub>3</sub>CO<sub>2</sub>H in CH<sub>3</sub>OH (v/v) (B).
Separation of nucleosides
involved the following gradient: 0 min (100% A, 0% B),
7
min (100% A, 0% B), 37 min (50%A, 50% B), 45 min (30%
A, 70% B), 55 min (100% A, 0% B), and 70 min (100% A,
0% B). The flow rate was 0.25 mL min<sup>-1</sup>.
Precursor ions
(the MH<sup>+</sup> ions of the DNA adducts) were generated in
the
ESI source and focused (quadrupole 1). These ions
were
dissociated in a collision cell (quadrupole 2), yielding
defined
product ions which were analyzed (quadrupole 3). The
optimal collisional offset voltage to maximize the yields
of
HO-ethanodGuo product ions was −18 mV.</p></sec></sec><sec id="d7e494"><title></title><sec id="d7e496"><title><italic toggle="yes">Synthesis of 1,N<sup>2</sup></italic><sup></sup>-ε-Gua-Modified
19-mer
</title><p><italic toggle="yes">5‘-O-(4,4‘-Dimethoxytrityl)-1,N<sup>2</sup>-ε-dGuo.</italic>
The dimethoxytritylation step was carried out as described in detail by
DeCorte et al. (1996) starting with 50 mg (0.17 mmol) of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo. Flash column chromatography
(CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH/pyridine, 98:1:1, v/v/v, isocratic) yielded 76
mg
(0.13 mmol, 74%) of
5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)-1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo: TLC <italic toggle="yes">R<sub>f</sub></italic> = 0.70
(CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH, 9:1, v/v); MS
<italic toggle="yes">m/z</italic>
(assignment and relative abundance in parentheses) 594
(MH<sup>+</sup>, 6), 303 (4,4‘-dimethoxytrityl<sup>+</sup>, 100),
176 [MH<sup>+</sup> −
dimethoxytrityldeoxyribose (ε-Gua<sup>+</sup>), 42];
<sup>1</sup>H-NMR
[(C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO] δ 1.90 (m, 1 H,
H-2‘), 2.21 (m, 1 H, H-2‘ ‘),
3.09 (m, 2 H, H-5‘, H-5‘ ‘), 3.40 (s, 1 H, H-4‘), 3.67 (s, 3
H,
C<italic toggle="yes">H</italic><sub>3</sub>O), 3.69 (s, 3 H,
C<italic toggle="yes">H</italic><sub>3</sub>O), 3.90 (m, 1 H, H-3‘), 5.75 (t,
1
H, H-1‘), 6.79−6.89 (m, 4 H, aromatic), 7.09−7.26 (m,
7
H, aromatic), 7.33−7.37 (m, 2 H, aromatic), 7.75 (d, 1
H,
H-6), 7.89 (d, 1 H, H-7), 8.57 (s, 1 H, H-2).
</p><p><italic toggle="yes">3‘-O-[(N,N-Diisopropylamino)(2-cyanoethyl)phosphinyl]-5‘-O-(4,4‘-dimethoxytrityl)-1,N<sup>2</sup>-ε-dGuo.</italic> The
synthesis of
the phosphoramidate derivative was achieved following
established procedures (Decorte et al., 1996), which were
modified by omitting the NaHCO<sub>3</sub> extraction step.
The
concentrated crude compound was instead directly subjected
to flash column chromatography (CH<sub>2</sub>Cl<sub>2</sub>/ethyl
acetate/pyridine, 69:30:1, v/v/v, isocratic), yielding 78 mg (0.098
mmol, 77%) of
3‘-<italic toggle="yes">O</italic>-[(<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropylamino)(2-cyanoethyl)phosphinyl-5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)-1,<italic toggle="yes">N</italic><italic toggle="yes"><sup>2</sup></italic><sup></sup>-ε-dGuo:
TLC <italic toggle="yes">R<sub>f</sub></italic>
= 0.72 (CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH, 9:1, v/v);
<sup>31</sup>P-NMR (C<sup>2</sup>H<sub>3</sub>CN) δ
149.42, 149.64. A signal at δ 15.52 (inorganic
phosphorus)
indicated ∼50% hydrolysis of the phosphoramidate.
Due
to this degradation, no further characterization of the
product
was performed and immediate oligonucleotide synthesis was
carried out.
</p></sec></sec><sec id="d7e648"><title></title><sec id="d7e650"><title>HO-ethanoGua-Modified 19-mer (Scheme <xref rid="bi962526vh00002"></xref>)
</title><p><italic toggle="yes">HO-ethanodGuo.</italic> HO-ethanoGua was
synthesized as
described elsewhere (Guengerich et al., 1993), and an
aliquot
was enzymatically converted to the nucleoside with
<italic toggle="yes">trans</italic>-<italic toggle="yes">N</italic>-deoxyribosylase (Müller et al., 1996) to serve as
a
chromatographic standard for the oligonucleotide digest
assay.
<fig id="bi962526vh00002" position="float" fig-type="scheme" orientation="portrait"><label>2</label><caption><p>Synthesis of Oligonucleotides Containing HO-ethanoGua<italic toggle="yes"><sup>a</sup></italic><sup></sup></p><p><fn id="d7e677"><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Ac<sub>2</sub>O =
(CH<sub>3</sub>)<sub>2</sub>CO; DMT Cl = 4,4‘-dimethoxytrityl
chloride; Block 3‘-OH indicates 2-cyanoethyl
<italic toggle="yes">N,N,N‘,N‘</italic>-tetraisopropylchlorophosphoramidite.</p></fn></p></caption><graphic xlink:href="bi962526vh00002.gif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">AcO-ethanoGua.</italic> HO-ethanoGua (25 mg, 0.13 mmol)
was
dried by repeated treatment with and evaporation of anhydrous pyridine <italic toggle="yes">in vacuo</italic> (3 × 2.5 mL). The compound
was
then redissolved in anhydrous pyridine (2 mL), and freshly
distilled (CH<sub>3</sub>CO)<sub>2</sub>O (0.061 mL, 0.65 mmol) was
added and
the mixture stirred for 3 h at room temperature under an
Ar
atmosphere. Most of the pyridine was removed <italic toggle="yes">in
vacuo</italic>
without heating by repeated addition of
hexane (3 × 2 mL).
The reddish-brown residue was dissolved in CH<sub>3</sub>OH
and
purified by HPLC. The peak eluting with a
<italic toggle="yes">t</italic><sub>R</sub> of 19.0 min
was collected, and the buffer salts were removed by
repeated
lyophilization to yield 29 mg (0.13 mmol, 95%) of
AcO-ethanoGua: MS <italic toggle="yes">m/z</italic> 236 (MH<sup>+</sup>, 58), 176
[MH<sup>+</sup> −
CH<sub>3</sub>CO − H<sub>2</sub>O (ε-Gua), 20];
<sup>1</sup>H-NMR [(C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO]
δ 2.03
(s, 3 H, C<italic toggle="yes">H</italic><sub>3</sub>CO), 3.51 (d, 1 H, H-6), 3.91 (dd, 1
H, H-6),
6.91 (d, 1 H, H-7), 7.68 (s, 1 H, NH), 8.14 (s, 1 H, H-2),
12.53 (s, 1 H, NH). Formation of the desired product
(as
compared to potential amide formation) was further confirmed by an alkaline hydrolysis experiment. Treatment
of
the product with NaOH (pH 9−10) at room temperature
resulted in immediate cleavage of the compound to the
starting material as demonstrated by HPLC analysis.
</p><p><italic toggle="yes">Deoxyribosylation of AcO-ethanoGua.</italic> The attachment
of
the deoxyribose to the protected DNA base adduct was
achieved by an enzymatic synthesis with
<italic toggle="yes">Lactobacillus
helveticus</italic> <italic toggle="yes">trans</italic>-<italic toggle="yes">N</italic>-deoxyribosylase following
a recently
described procedure (Müller et al., 1996).
AcO-ethanoGua
(29 mg, 0.13 mmol) was converted to tetrahydro-7-acetoxy-9-oxo-3-β-<sc>d</sc>-deoxyribofuranosylimidazo[1,2-<italic toggle="yes">a</italic>]purine
(diastereomers <bold>1</bold> and <bold>2</bold>) (39 mg, 0.11 mmol, 90%
combined
yield). As expected, a set of two diastereomers due to
the
presence of two chiral centers in the molecule (H-7 and
H-1‘)
was detected upon HPLC analysis, occurring in a 1:1 ratio.
The two peaks eluting with a <italic toggle="yes">t</italic><sub>R</sub> of 20.2 min
(diastereomer
<bold>1</bold>) and 20.8 min (diastereomer <bold>2</bold>) were collected
and
characterized separately. Diastereomer <bold>1</bold>: MS
<italic toggle="yes">m/z</italic> 352
(MH<sup>+</sup>, 6), 337 (MH<sup>+</sup> − CH<sub>3</sub>,
32), 236 (MH<sup>+</sup> − deoxyribose,
8), 176 [MH<sup>+</sup> − deoxyribose − CH<sub>3</sub>CO −
H<sub>2</sub>O (ε-Gua<sup>+</sup>),
6]; <sup>1</sup>H-NMR (<sup>2</sup>H<sub>2</sub>O) δ 2.14 (s, 3
H, C<italic toggle="yes">H</italic><sub>3</sub>CO), 2.51 (m, 1 H,
H-2‘), 2.77 (m, 1 H, H-2‘ ‘), 3.78 (m, 3 H, H-5‘, H-5‘ ‘,
H-6),
4.09 (m, 2 H, H-4‘, H-6), 4.61 (m, 1 H, H-3‘), 6.29 (t, 1
H,
H-1‘), 7.10 (d, 1 H, H-7), 7.98 (s, 1 H, H-2).
Diastereomer
<bold>2</bold>: MS <italic toggle="yes">m/z</italic> 352 (MH<sup>+</sup>, 44), 236
(MH<sup>+</sup> − deoxyribose, 62);
<sup>1</sup>H-NMR (<sup>2</sup>H<sub>2</sub>O) δ 2.12 (s, 3 H,
C<italic toggle="yes">H</italic><sub>3</sub>CO), 2.49 (m, 1 H,
H-2‘), 2.73 (m, 1 H, H-2‘ ‘), 3.75 (m, 3 H, H-5‘, H-5‘ ‘,
H-6),
4.06 (m, 2 H, H-4‘, H-6), 4.58 (m, 1 H, H-3‘), 6.26 (t, 1
H,
H-1‘), 7.07 (d, 1 H, H-7), 7.96 (s, 1 H, H-2). While
the
above analytical data allowed no distinction between the
two
diastereomers, the antiphasic orientation of the CD
spectra
clearly demonstrated the postulated stereochemistry
(<italic toggle="yes">vide
infra</italic>).
</p><p><italic toggle="yes">5‘-O-(4,4‘-Dimethoxytrityl)-5,6,7,9-tetrahydro-7-acetoxy-9-oxo-3-β-<sc>d</sc>-deoxyribofuranosylimidazo[1,2-a]purine
(Diastereomers</italic> <bold><italic toggle="yes">1</italic></bold> <italic toggle="yes">and</italic> <bold><italic toggle="yes">2</italic></bold><italic toggle="yes">).</italic>
The dimethoxytritylation was done
with the combined materials from the previous step (39 mg,
0.11 mmol) following the established protocol (Decorte et
al., 1996) with a modification in the extraction step.
The
10% K<sub>2</sub>CO<sub>3</sub> solution was replaced by
H<sub>2</sub>O to avoid hydrolysis of the acetyl ester. Flash column chromatography
(ethyl
acetate/CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH/pyridine,
55:45:0.1:0.2, v/v/v/v, isocratic) yielded 54 mg (0.083 mmol, 75%) of
5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)-5,6,7,9-tetrahydro-7-acetoxy-9-oxo-3-β-<sc>d</sc>-deoxyribofuranosylimidazo[1,2-<italic toggle="yes">a</italic>]purine (diastereomers
<bold>1</bold>
and <bold>2</bold>): TLC <italic toggle="yes">R<sub>f</sub></italic> = 0.61 and 0.71
(ethyl acetate/CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH, 50:50:0.2, v/v/v); MS <italic toggle="yes">m/z</italic> 654
(MH<sup>+</sup>, 6), 303 (4,4‘-dimethoxytrityl<sup>+</sup>, 100); <sup>1</sup>H-NMR
[(C<sup>2</sup>H<sub>3</sub>)<sub>2</sub>SO] δ 1.71 (m,
1
H, H-2‘), 1.87 (m, 1 H, H-2‘ ‘), 2.13 (s, 3 H,
C<italic toggle="yes">H</italic><sub>3</sub>CO), 2.79
(m, 3 H, H-5‘, H-5‘ ‘, H-6), 3.05 (m, 2 H, H-4‘, H-6), 3.70
(s, 3 H, C<italic toggle="yes">H</italic><sub>3</sub>O), 3.72 (s, 3 H,
C<italic toggle="yes">H</italic><sub>3</sub>O), 4.06 (m, 1 H, H-3‘),
5.62 (t, 1 H, H-1‘), 6.67−6.82 (m, 4 H, aromatic), 6.90
(d,
1 H, H-7), 7.12−7.30 (m, 7 H, aromatic), 7.31−7.44 (m,
2
H, aromatic), 7.50 (s, 1 H, H-2).
</p><p><italic toggle="yes">3‘-O-[(N,N-Diisopropylamino)(2-cyanoethyl)phosphinyl]-5‘-O-(4,4‘-dimethoxytrityl)-5,6,7,9-tetrahydro-7-acetoxy-9-oxo-3-β-<sc>d</sc>-deoxyribofuranosylimidazo[1,2-a]purine
(Diastereomers</italic> <bold><italic toggle="yes">1</italic></bold> <italic toggle="yes">and</italic> <bold><italic toggle="yes">2</italic></bold><italic toggle="yes">).</italic>
The phosphoramidation step followed
the procedure used for the 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo
derivative. Direct
application of the crude product to flash column chromatography (CH<sub>2</sub>Cl<sub>2</sub>/ethyl acetate/pyridine,
69:30:1, v/v/v,
isocratic) yielded 56 mg (0.066 mmol, 80%) of
3‘-<italic toggle="yes">O</italic>-[(<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropylamino)(2-cyanoethyl)phosphinyl]-5‘-<italic toggle="yes">O</italic>-(4,4‘-dimethoxytrityl)-5,6,7,9-tetrahydro-7-acetoxy-9-oxo-3-β-<sc>d</sc>-deoxyribofuranosylimidazo[1,2-<italic toggle="yes">a</italic>]purine (diastereomers
<bold>1</bold>
and <bold>2</bold>): TLC <italic toggle="yes">R<sub>f</sub></italic> = 0.60 (ethyl
acetate/CH<sub>2</sub>Cl<sub>2</sub>/CH<sub>3</sub>OH,
50:49.8:0.2, v/v/v); <sup>31</sup>P-NMR (C<sup>2</sup>H<sub>3</sub>CN)
δ 149.62, 149.72.
Again, a signal at δ 15.53 (inorganic phosphorus)
indicated
∼50% hydrolysis of the product. Thus further
characterization was not done in favor of immediate oligonucleotide
synthesis.
</p></sec></sec><sec id="d7e1062"><title></title><sec id="d7e1064"><title>Oligonucleotides (Scheme <xref rid="bi962526vh00003"></xref>)
</title><p>Oligonucleotides were synthesized with an
Expedite
Nucleic Acid Synthesis System (Millipore Corp., Bedford,
MA) on a 1 μmol scale using
4-<italic toggle="yes">tert</italic>-butylphenoxyacetyl
protecting groups (PerSeptive Biosystems) according to the
manufacturer‘s standard protocol. To compensate for
the
hydrolysis of the phosphoramidate, the DNA adduct nucleosides were dissolved, making them twice as concentrated
(100 mg mL<sup>-1</sup>) as the unmodified nucleosides (50 mg
mL<sup>-1</sup>).
Nevertheless, a drop of 50% in overall coupling
efficiency
was noted after the insertion of the respective DNA
adducts
into the sequences in each synthesis. Following
synthesis,
the beads from two 1 μmol cassettes of each DNA oligomer
were suspended in 50 mM NaOH (3 mL) and stirred slowly
for 24 h at room temperature. After cautious
neutralization
with 50 mM CH<sub>3</sub>CO<sub>2</sub>H (1 mL), the solutions were
filtered
through a 0.22 μm filter and aliquots were subjected to
HPLC
analysis and preparative workup. The unmodified
19-mer
and the 10-mer primer (Scheme <xref rid="bi962526vh00003"></xref>) each showed one major
peak with a <italic toggle="yes">t</italic><sub>R</sub> of 49.0 and 54.2 min,
respectively, which
were collected and repeatedly lyophilized to remove buffer
salts. Yields after the initial purification were 39 and
70
<italic toggle="yes">A</italic><sub>260</sub> units [0.21 and 0.70 μmol, respectively,
estimated by
the method of Borer (1975)]. HPLC analysis of the
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua-modified 19-mer demonstrated two peaks with
<italic toggle="yes">t</italic><sub>R</sub>s of
49.7 and 50.2 min in a 1:1 ratio, which were purified
separately. Upon enzymatic digestion, the second peak
was
identified to contain the DNA adduct, and a yield of 38
<italic toggle="yes">A</italic><sub>260</sub>
units (∼0.2 μmol) was determined. The
OH-ethanoGua-modified 19-mer was also a mixture of two product peaks,
eluting with <italic toggle="yes">t</italic><sub>R</sub>s of 50.5 and 51.5 min in a ratio
of 3:7. HPLC
analysis of the enzymatic digest of the oligomer revealed
that the first product peak contained the modified
nucleoside;
its yield was 19 <italic toggle="yes">A</italic><sub>260</sub> units (∼0.1 μmol).
All DNA oligomers
were subjected to a second HPLC purification and further
polyacrylamide gel electrophoresis to
obtain purities of
>99% as evaluated by CGE (<italic toggle="yes">vide infra</italic>).
<fig id="bi962526vh00003" position="float" fig-type="scheme" orientation="portrait"><label>3</label><caption><p>Oligonucleotides<sup><italic toggle="yes">a</italic></sup></p><p><fn id="d7e1138"><p><sup><italic toggle="yes">a</italic></sup> G* = Gua,
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, or HO-ethanoGua. The site of the
first
incorporation is indicated with an arrow.</p></fn></p></caption><graphic xlink:href="bi962526vh00003.gif" position="float" orientation="portrait"></graphic></fig></p></sec></sec><sec id="d7e1152"><title></title><sec id="d7e1154"><title>Characterization of Oligonucleotides
</title><p>Direct injection electrospray mass spectrometry was
used
to verify the identities of three of the four oligomers
used:
10-mer, calcd <italic toggle="yes">M</italic><sub>r</sub> of 3027.0, anal. 3026.9;
unmodified 19-mer, calcd <italic toggle="yes">M</italic><sub>r</sub> of 5883.9, anal. 5884.9; and
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua 19-mer, calcd <italic toggle="yes">M</italic><sub>r</sub> of 5907.9, anal. 5908.3 (see
Supporting
Information). Attempts to analyze the intact
HO-ethanoGua-containing 19-mer directly were unsuccessful.
</p><p>Each oligonucleotide (1 <italic toggle="yes">A</italic><sub>260</sub> unit =
∼5.4 nmol) was
digested in a two-step protocol. The first step
involved
dissolving the DNA oligomer in 20 μL of 10 mM Tris-HCl
buffer (pH 7.0) containing 10 mM MgCl<sub>2</sub> and the
addition
of 8 μg of nuclease P<sub>1</sub> (Sigma Chemical Co., St. Louis,
MO),
followed by a 3 h incubation at 37 °C. In the second
step,
20 μL of 100 mM Tris-HCl buffer (pH 9.0), 9 μg of
snake
venom phosphodiesterase (Sigma), and 6 μg of alkaline
phosphatase (Sigma) were added and the digest was kept
for another 3 h at 37 °C. Each sample was diluted with
100
μL of H<sub>2</sub>O and filtered through a 0.22 μm filter; a 50
μL
aliquot was analyzed by HPLC, and the concentrations of
the individual deoxyribonucleosides were estimated by
comparisons made with external standards: 10-mer primer
(Scheme <xref rid="bi962526vh00003"></xref>), theoretical (relative ratios in parentheses)
dCyd
(2), dGuo (2), dThd (3), dAdo (3) and found dCyd (2.0),
dGuo (2.4), dThd (3.3), dAdo (3.4); unmodified 19-mer
template, theoretical dCyd (3), dGuo (7) dThd (5), dAdo
(4)
and found dCyd (3.0), dGuo (7.2), dThd (5.3), dAdo (4.2);
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua-containing 19-mer template,
theoretical dCyd (3),
dGuo (6), dThd (5), dAdo (4), 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo (1)
and found
dCyd (3.0), dGuo (6.1), dThd (5.3), dAdo (4.2),
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo
(1.0). In the case of the HO-ethanoGua-containing
19-mer
template, the OH-ethanodGuo and dThd were not resolved
by HPLC, and the sample (<sup>1</sup>/<sub>10</sub> the normal amount
was
digested) was analyzed by combined HPLC/ES MS/MS
(Müller et al., 1997). HO-ethanodGuo was identified
and
confirmed by repeating the analysis in the presence of a
known amount of 4,5,8-<sup>13</sup>C-labeled compound (Müller et
al.,
1997) (<italic toggle="yes">vide infra</italic>).
</p></sec></sec><sec id="d7e1225"><title></title><sec id="d7e1227"><title>Enzymes
</title><p><italic toggle="yes">trans</italic>-<italic toggle="yes">N</italic>-Deoxyribosylase was
partially purified from <italic toggle="yes">L.
helveticus</italic> (Müller et al., 1996) by L. K. Hutchinson in
the
Department of Biochemistry at the Vanderbilt University
School of Medicine. Recombinant rat pol β was a gift of
S.
Wilson (University Texas, Galveston, TX). Other polymerases were purified by L. L. Furge from <italic toggle="yes">Escherichia
coli</italic>
(Lowe & Guengerich, 1996) using stock plasmids provided: Kf (C. Joyce, Yale University, New Haven, CT)
(Derbyshire et al., 1988), pol II (M. F. Goodman,
University
of Southern California, Los Angeles, CA) (Cai et al.,
1995),
pol T7 and thioredoxin (K. A. Johnson, Pennsylvania State
University, University Park, PA) (Kati et al., 1991; Lunn
et
al., 1984), and RT (S. Hughes, Frederick Cancer Facility,
Frederick, MD) (Le Grice & Grüninger-Leitch, 1990).
</p></sec></sec><sec id="d7e1243"><title></title><sec id="d7e1245"><title>Polymerase Assays
</title><p><italic toggle="yes">General.</italic> The 10-mer primer (2 μM)
was 5‘-end-labeled
using T4 polynucleotide kinase and purified on a Biospin
column (Bio-Rad, Hercules, CA). Template and labeled
primer (2:1 molar ratio) were annealed in a buffer
containing
50 mM sodium MOPS (pH 7.0), 50 μg of bovine serum
albumin per milliliter, and 5 mM MgCl<sub>2</sub> by incubating
at
90 °C for 10 min and slowly cooling to room temperature.
The different assays were then performed as follows.
</p><p><italic toggle="yes">Primer Extension.</italic> All four dNTPs, at 100 μM
each, were
incubated in the presence of 50 or 100 nM primer/template
mixture in 10 μL of 50 mM sodium MOPS buffer (pH 7.0)
containing 8 mM MgCl<sub>2</sub>, 4 mM dithiothreitol, 2 μg of
bovine
serum albumin per milliliter, and the particular
polymerase,
added at several different concentrations. These
reactions
were performed for 30 min at 25 °C with all polymerases,
except for pol II which was incubated at 37 °C.
Reactions
were quenched by the addition of 10 mM EDTA in 90%
formamide (v/v), and the reaction products were analyzed
by electrophoresis on 20% (w/v) denaturating
polyacrylamide
gels using Sequagel (National Diagnostics, Atlanta, GA).
</p><p><italic toggle="yes">One-Base Incorporation.</italic><sup>32</sup>P-labeled 10-mer primers were
extended using unmodified or adducted 19-mer templates
in the presence of single dNTPs (100 μM) with 50 nM Kf
or pol II, 200 nM T7, or 4.6 μM pol β. The incubation
times
were 15 min for pol I and pol II, but in the case of T7
and
pol β, the reaction time was extended to 30 min.
</p><p><italic toggle="yes">Steady-State Kinetics.</italic> The general approach of
Boosalis
et al. (1987) was used, as modified in this laboratory
(Lowe
& Guengerich, 1996). A Molecular Dynamics Model 400E
Phosphorimager (Molecular Dynamics Inc., Sunnyvale, CA)
was used to measure incorporation of radioactivity, and
the
results were analyzed using a k·cat computer program
(Biometallics, Princeton, NJ) as described elsewhere (Lowe
& Guengerich, 1996).
</p><p><italic toggle="yes">Nucleotide Sequence Analysis.</italic> The bands
corresponding
to 100 pmol of the primer extension products obtained with
the several enzymes were extracted from the gel by shaking
overnight at 4 °C in distilled H<sub>2</sub>O. The products
were
recovered by C<sub>2</sub>H<sub>5</sub>OH precipitation and sequenced
as
described by Maxam and Gilbert (1980) except for the
T-specific reaction, where the method described by Friedmann and Brown (1978) was used.
</p></sec></sec><sec id="d7e1286"><title>Results</title><p><italic toggle="yes">Synthesis of Protected HO-ethanodGuo.</italic>
The preparation
of an oligonucleotide substituted with HO-ethanoGua required development of an appropriate strategy for
incorporation of a suitably substituted phosphoramidite (Scheme <xref rid="bi962526vh00002"></xref>).
The free hydroxyl group on the exocyclic ring should
be
blocked in order to prevent coupling in the
phosphoramidite
reaction of the DNA synthesizer, using a group that can be
readily deprotected. The hydroxyl group was readily
acetylated in pyridine. The deoxyribose moiety could be
enzymatically added to HO-ethanoGua with purine nucleoside
phosphorylase, but the acetylated derivative gave no
nucleoside product with this enzyme. However, <italic toggle="yes">L. helveticus</italic><italic toggle="yes">trans-N</italic>-deoxyribosylase catalyzed the reaction and was used
in
the synthesis (Müller et al., 1996).<xref rid="atyp_ref2" ref-type="bibr"></xref></p><p>HPLC of the acetylated nucleoside revealed two
closely
eluting peaks with similar areas. These two compounds
had
identical UV, mass, and <sup>1</sup>H-NMR spectra. However, the
CD
spectra were mirror images of each other (see Supporting
Information), supporting the conclusion
that these are diastereomers and differ in their configuration at C7.
The
absolute configurations of these diastereomers were not
determined. Treatment with 0.10 N NaOH led to the
loss
of the CD spectra at wavelengths of >250 nm, and a single
HPLC peak was eluted.
<fig id="bi962526vf00001" position="float" orientation="portrait"><label>1</label><caption><p>CGE traces of oligonucleotides: (A) primer (10-mer), (B) 19-mer template (unmodified), (C) 19-mer containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, and (D) 19-mer containing HO-ethanoGua.</p></caption><graphic xlink:href="bi962526vf00001.gif" position="float" orientation="portrait"></graphic></fig></p><p>The evidence for the diastereomers raised the issue
of
whether individual hydroxyl diastereomers exist and could
be used separately. Alternatively, the closed and open
forms,
HO-ethanoGua and <italic toggle="yes">N</italic><sup>2</sup>-(2-oxoethyl)Gua,
respectively, might
be in equilibrium and the hydroxyl group would epimerize
(Scheme <xref rid="bi962526vh00001"></xref>). In order to test this possibility, the
exchange
of oxygen from H<sub>2</sub><sup>18</sup>O was examined with
HO-ethanodGuo,
using FAB MS and measuring the intensity of the ions at
<italic toggle="yes">m/z</italic> 310 and 312. At neutral pH, 72% of the
HO-ethanodGuo
had incorporated one <sup>18</sup>O atom following overnight
incubation at room temperature. When the experiment was
repeated
in 0.10 N NaOH, conditions to be used in deblocking after
oligonucleotide synthesis, the nucleoside had completely
incorporated one <sup>18</sup>O atom (>95% incorporation).
Thus,
HO-ethanoGua appears to be in relatively rapid equilibrium
between the open and the two (stereochemically different)
closed forms.
</p><p><italic toggle="yes">Synthesis and Characterization of Oligonucleotides.</italic>
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo and the acetoxy-protected form of HO-ethanodGuo
prepared above were treated to add 5‘-dimethoxytrityl and
3‘-phosphoramidite groups in the usual manner and used to
prepare 19-mer oligonucleotides (Scheme <xref rid="bi962526vh00002"></xref>). The
coupling
yields at the site of the derivative were 50% of those at
the
other steps, even with excess derivatized
phosphoramidites.
Deblocking of the HO-ethanoGua derivative was done in
0.05 N NaOH instead of NH<sub>4</sub>OH to remove the
acetoxy
group. The 19-mer templates and the 10-mer primer
were
purified by a combination of reversed-phase HPLC and
preparative polyacrylamide gel electrophoresis, taking
care
to slice only the middle portion of the gel band in the
more
demanding separations.
</p><p>CGE electrophoretograms of the purified oligomers
are
shown (Figure <xref rid="bi962526vf00001"></xref>). Purity in all cases was judged to
be
>99%.
</p><p>The oligomers containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
and HO-ethanoGua
were digested with nucleases and phosphatase, and the
products were analyzed by HPLC (Figure <xref rid="bi962526vf00002"></xref>). The
distinct
UV spectra and <italic toggle="yes">t</italic><sub>R</sub> permitted positive
identification of the
former oligomer, using diode array spectroscopy, to characterize 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo (Figure <xref rid="bi962526vf00002"></xref>A). With
HO-ethanodGuo,
the similarity of the <italic toggle="yes">t</italic><sub>R</sub> to that of dThd and the
lack of such
a distinct spectrum precluded such analysis. The
nucleoside/phosphatase digest of this oligomer was analyzed by HPLC/ES MS/MS as reported elsewhere (Müller et al., 1997).
The
presence of HO-ethanoGua was clearly verified by monitoring the appropriate transition (Figure <xref rid="bi962526vf00002"></xref>B).
<fig id="bi962526vf00002" position="float" orientation="portrait"><label>2</label><caption><p>Analysis of digests of modified oligomers. See Experimental Procedures for description. (A) HPLC/UV of the digest of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua-containing 19-mer, with
the spectrum of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-dGuo in the inset. (B) HPLC/ES MS/MS of the digest of
HO-ethanoGua-containing 19-mer, with <italic toggle="yes">m/z</italic> traces shown for
the
appropriate MS/MS transitions of dAdo, dCyd, dGuo, dThd,
HO-ethanodGuo, and [4,5,8-<sup>13</sup>C]HO-ethanodGuo (heavy atom
derivative
added to verify <italic toggle="yes">t</italic><sub>R</sub>) (Müller et al., 1997).
HPLC/selected reaction
monitoring/MS/MS was done monitoring the 310 to 194 or the
313
to 197 transition (loss of deoxyribose) in the latter two cases.
No
<italic toggle="yes">m/z</italic> 313 transition was seen in the absence of added
<sup>13</sup>C material,
but the same <italic toggle="yes">m/z</italic> 310 transition peak was seen.</p></caption><graphic xlink:href="bi962526vf00002.gif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Polymerase Extension Assays.</italic> Five polymerases
were
studied because of their availability and existing
literature
regarding their properties. The initial experiments
involved
analysis of the extension of the 10-mer primer/19-mer
template complexes in the presence of a mixture of all
four
dNTPs. With all five polymerases, both of the
modified
templates retarded extension but some full-length product
was obtained (Figures
<xref rid="bi962526vf00003"></xref>−7).
The results are qualitatively
summarized in Table <xref rid="bi962526vt00001"></xref>.
<fig id="bi962526vf00003" position="float" orientation="portrait"><label>3</label><caption><p>Extension of 10-mer primer by Kf in the presence of all four dNTPs.</p></caption><graphic xlink:href="bi962526vf00003.gif" position="float" orientation="portrait"></graphic></fig><fig id="bi962526vf00004" position="float" orientation="portrait"><label>4</label><caption><p>Extension of 10-mer primer by pol II in the presence of all four dNTPs.</p></caption><graphic xlink:href="bi962526vf00004.gif" position="float" orientation="portrait"></graphic></fig><fig id="bi962526vf00005" position="float" orientation="portrait"><label>5</label><caption><p>Extension of 10-mer primer by T7 pol in the presence of all four dNTPs.</p></caption><graphic xlink:href="bi962526vf00005.gif" position="float" orientation="portrait"></graphic></fig><fig id="bi962526vf00006" position="float" orientation="portrait"><label>6</label><caption><p>Extension of 10-mer primer by RT in the presence of all four dNTPs.</p></caption><graphic xlink:href="bi962526vf00006.gif" position="float" orientation="portrait"></graphic></fig><fig id="bi962526vf00007" position="float" orientation="portrait"><label>7</label><caption><p>Extension of 10-mer primer by pol β in the presence of all four dNTPs.</p></caption><graphic xlink:href="bi962526vf00007.gif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi962526vt00001" position="float" orientation="portrait"><label>1</label><caption><p>Products of Polymerase Extension Assays of 10-mer Primer in the Presence of All Four dNTPs and a 19-mer Template<italic toggle="yes"><sup>a</sup></italic><sup></sup></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 namest="2" nameend="4">products from each template (length)</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">polymerase</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">HO-ethanoGua
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Kf
</oasis:entry><oasis:entry colname="2">19, 20
</oasis:entry><oasis:entry colname="3">11, 17−20
</oasis:entry><oasis:entry colname="4">11, 19, 20 (plus 12−18)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol II
</oasis:entry><oasis:entry colname="2">18, 19
</oasis:entry><oasis:entry colname="3">18, 19
</oasis:entry><oasis:entry colname="4">18, 19 (plus 11−17)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T7
</oasis:entry><oasis:entry colname="2">19, 20
</oasis:entry><oasis:entry colname="3">18−20
</oasis:entry><oasis:entry colname="4">19, 20 (plus 11−18)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">RT
</oasis:entry><oasis:entry colname="2">18, 19
</oasis:entry><oasis:entry colname="3">20, 9−7
</oasis:entry><oasis:entry colname="4">20, 9−7
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol β
</oasis:entry><oasis:entry colname="2">19
</oasis:entry><oasis:entry colname="3">19
</oasis:entry><oasis:entry colname="4">19</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> See Scheme <xref rid="bi962526vh00003"></xref> and Figures
<xref rid="bi962526vf00003"></xref>−7.</p></table-wrap-foot></table-wrap></p><p>With all of the polymerases except pol β, extension
of
the HO-ethanoGua-containing 19-mer yielded a ladder of all
the possible products differing in length by one base.
Kf
also yielded a large amount of 11-mer with both modified
oligomers, corresponding to addition of only one base at
the
site of the modified Gua. Under these conditions, Kf
often
extends a primer one base beyond the
length of the template
(Clark et al., 1987; Clark, 1988), and this phenomenon was
observed with all three templates with Kf, T7, and RT
(Figures <xref rid="bi962526vf00003"></xref>, <xref rid="bi962526vf00005"></xref>, and 6). Although RT is usually considered
to
be devoid of exonuclease activity (Kornberg & Baker,
1992),
digestion of this sequence was observed, and the extent
was
increased when the modified bases were present (Figure <xref rid="bi962526vf00006"></xref>).
This appears not to be a sequence-dependent activity,
because
we also observed such digestion with another oligomer used
previously in other work (Lowe & Guengerich, 1996) and
inspection of other work indicates similar RT processing
(Kati et al., 1992).
</p><p><italic toggle="yes">Nucleotide Sequence Analysis of Extended Primers.</italic>
The
products obtained by primer extension in the presence of
all
four dNTPs were analyzed using modifications of the
Maxam−Gilbert method (Maxam & Gilbert, 1980), and the
results are summarized in Table <xref rid="bi962526vt00002"></xref>. The extent of
misincorporation cannot be analyzed quantitatively in such assays,
and the results must be considered qualitatively.
Typical
gels obtained in the analysis of the Kf products with the
modified bases are shown in Figure <xref rid="bi962526vf00008"></xref>.
Products from the
reactions with RT and pol β were not analyzed.
<fig id="bi962526vf00008" position="float" orientation="portrait"><label>8</label><caption><p>Nucleotide sequence analysis. (A) Residues 9−13 of standard 19-mers synthesized to contain A, C, G, and T at position 11. Also shown are the (B) 11-mer, (C) 19-mer, and (D) 17-mer formed by extension when paired to the 19-mer template containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua.</p></caption><graphic xlink:href="bi962526vf00008.gif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi962526vt00002" position="float" orientation="portrait"><label>2</label><caption><p>Nucleotide Sequence Analysis of a 10-mer Primer Extended in the Presence of All Four dNTPs and 19-mer Primer</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">bases incorporated opposite
Gua or Gua adduct</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">polymerase</oasis:entry><oasis:entry colname="2">length of
product</oasis:entry><oasis:entry colname="3">Gua</oasis:entry><oasis:entry colname="4">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua</oasis:entry><oasis:entry colname="5">HO-ethanoGua
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Kf
</oasis:entry><oasis:entry colname="2">19,<italic toggle="yes"><sup>a</sup></italic><sup></sup> 20
</oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">G, C (A) <italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5">G, T (A)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">18</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4">−1 base frameshift</oasis:entry><oasis:entry colname="5">−1 base frameshift</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">17<sup><italic toggle="yes">a</italic></sup></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4">−2 base frameshift</oasis:entry><oasis:entry colname="5">−2 base frameshift</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">11</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4">A, G</oasis:entry><oasis:entry colname="5">A, G
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol II
</oasis:entry><oasis:entry colname="2">19
</oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">A, T (C)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5">A, T (C)<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">18</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4">−1 base frameshift</oasis:entry><oasis:entry colname="5">−1 base frameshift
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T7
</oasis:entry><oasis:entry colname="2">19, 20
</oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">C
</oasis:entry><oasis:entry colname="5">C</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">18</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4">−1 base frameshift</oasis:entry><oasis:entry colname="5">−1 base frameshift</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> See Figure
<xref rid="bi962526vf00008"></xref>.<sup><italic toggle="yes">b</italic></sup> Tentative assignment. See Figure <xref rid="bi962526vf00008"></xref>
for data in the
case of 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and Kf. See Figure <xref rid="bi962526vf00009"></xref> and
Tables <xref rid="bi962526vt00003"></xref> and <xref rid="bi962526vt00004"></xref> for single-base incorporation results.</p></table-wrap-foot></table-wrap></p><p>Kf and pol II yielded more misincorporation than
T7.
Differences between the products derived with the
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-
Gua- and HO-ethanoGua-containing
oligomers were seen.
Also, there were differences in the bases misincorporated
in
the 11-mer (one-base incorporation) and the fully extended
primer with Kf. In the case of the 11-mer product
formed
with Kf, both A and G were clearly present (Figure <xref rid="bi962526vf00008"></xref>B).
In
the 19-mer Kf product, G was clearly inserted (Figure <xref rid="bi962526vf00008"></xref>C)
but the relative intensities of bands in the G and G + A
lanes, compared with those of the standard oligomers
(Figure
<xref rid="bi962526vf00008"></xref>A), did not allow an unambiguous call for A. Similarly,
a
definite call for C in the case of the 19-mer product of
pol
II was not possible because of the insertion of T and the
relative band intensities in the T and T + C lanes.
</p><p><italic toggle="yes">Primer Extension Assays with Individual dNTPs.</italic>
The
incorporation assays were repeated to incorporate only a
single base; i.e., only use one dNTP in the assay. In
all
cases, it was possible to force some misincorporation
(Figure
<xref rid="bi962526vf00009"></xref>), even with the unmodified oligomer. Following
incorporation of a single base, the next position in the
template
contains a T, so incorporation of an A is then favored.
In
most cases in which dATP was used, some 12-mer was
found.
<fig id="bi962526vf00009" position="float" orientation="portrait"><label>9</label><caption><p>Extension of primer in the presence of a single dNTP when paired with modified 19-mers: (A) Kf, (B) pol II, (C) T7, and (D) pol β.</p></caption><graphic xlink:href="bi962526vf00009.gif" position="float" orientation="portrait"></graphic></fig></p><p>The results are summarized in Table <xref rid="bi962526vt00003"></xref> and are
considered
qualitative. Of interest is the observation of the
13-mer
product in the reaction of the 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
template with Kf
(Figure <xref rid="bi962526vf00009"></xref>A). Incorporation of three Cs at sites 11−13
is
highly unlikely, since pairing of dCTP opposite a T would
have to occur after the incorporation of dCTP opposite
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua (Scheme <xref rid="bi962526vh00003"></xref>). Moreover, further incorporation of
more
dCTP would have been expected. An alternative is that
the
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and the following T slip and the
three Cs are
incorporated opposite the G<sub>3</sub> triplet at positions
13−15
(Scheme <xref rid="bi962526vh00003"></xref>). Sequence analysis indicated that this
latter
slippage event occurred (Figure <xref rid="bi962526vf00008"></xref>D).
<table-wrap id="bi962526vt00003" position="float" orientation="portrait"><label>3</label><caption><p>Products of Polymerase Extension Assays of 10-mer Primers in the Presence of Modified 19-mers and a Single dNTP</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 namest="2" nameend="4">products from each template<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">polymerase</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">HO-ethanoGua
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Kf
</oasis:entry><oasis:entry colname="2">C ≫ A, G, T
</oasis:entry><oasis:entry colname="3">A > C > G
</oasis:entry><oasis:entry colname="4">A > C > G
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol II
</oasis:entry><oasis:entry colname="2">C ≫ A, G, T
</oasis:entry><oasis:entry colname="3">A > C
</oasis:entry><oasis:entry colname="4">A > C
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T7
</oasis:entry><oasis:entry colname="2">C
</oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">A > C
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pol β
</oasis:entry><oasis:entry colname="2">C
</oasis:entry><oasis:entry colname="3">C
</oasis:entry><oasis:entry colname="4">A > C</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> See Figure <xref rid="bi962526vf00009"></xref>.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Steady-State Kinetic Assays of Single-Base
Incorporation.</italic>
Single dNTP incorporation assays were done under
typical
steady-state conditions (Boosalis et al., 1987) in order
to
provide more quantitative estimates of the tendency of
polymerases to misincorporate bases. The results are
summarized in Table <xref rid="bi962526vt00004"></xref>.
<table-wrap id="bi962526vt00004" position="float" orientation="portrait"><label>4</label><caption><p>Steady-State Kinetic Parameters for dNTP Incorporation</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="7"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry namest="2" nameend="4">Kf</oasis:entry><oasis:entry namest="5" nameend="7">T7</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">pairing</oasis:entry><oasis:entry colname="2"><italic toggle="yes">k</italic><sub>cat</sub> (min<sup>-1</sup>)</oasis:entry><oasis:entry colname="3"><italic toggle="yes">K</italic><sub>m</sub> (μM)</oasis:entry><oasis:entry colname="4">misincorporation
frequency<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5"><italic toggle="yes">k</italic><sub>cat</sub> (min<sup>-1</sup>)</oasis:entry><oasis:entry colname="6"><italic toggle="yes">K</italic><sub>m</sub> (μM)</oasis:entry><oasis:entry colname="7">misincorporation
frequency
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Gua·dCTP
</oasis:entry><oasis:entry colname="2">81 ± 5
</oasis:entry><oasis:entry colname="3">0.027 ± 0.008
</oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">0.15 ± 0.01
</oasis:entry><oasis:entry colname="6">1.8 ± 0.7</oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua·dCTP
</oasis:entry><oasis:entry colname="2">0.54 ± 0.02
</oasis:entry><oasis:entry colname="3">67 ± 8</oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">0.0046 ± 0.0002
</oasis:entry><oasis:entry colname="6">1.1 ± 0.3</oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua·dGTP
</oasis:entry><oasis:entry colname="2">0.27 ± 0.02
</oasis:entry><oasis:entry colname="3">31 ± 8
</oasis:entry><oasis:entry colname="4">1.1<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua·dATP
</oasis:entry><oasis:entry colname="2">0.12 ± 0.01
</oasis:entry><oasis:entry colname="3">75 ± 24
</oasis:entry><oasis:entry colname="4">0.2<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua·dCTP
</oasis:entry><oasis:entry colname="2">0.28 ± 0.02
</oasis:entry><oasis:entry colname="3">5 ± 2</oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">0.0032 ± 0.002
</oasis:entry><oasis:entry colname="6">0.48 ± 0.17</oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua·dGTP
</oasis:entry><oasis:entry colname="2">0.35 ± 0.01
</oasis:entry><oasis:entry colname="3">41 ± 7
</oasis:entry><oasis:entry colname="4">0.15</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HO-ethanoGua·dATP
</oasis:entry><oasis:entry colname="2">0.17 ± 0.01
</oasis:entry><oasis:entry colname="3">7 ± 3
</oasis:entry><oasis:entry colname="4">0.45
</oasis:entry><oasis:entry colname="5">0.0052 ± 0.0003
</oasis:entry><oasis:entry colname="6">0.94 ± 0.33
</oasis:entry><oasis:entry colname="7">1.2</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Misincorporation frequency =
(<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).<sup><italic toggle="yes">b</italic></sup> These are
considered to be much higher because the values
for dCTP incorporation reflect incorporation of three dCTPs. See the
text for discussion of a possible frameshift.</p></table-wrap-foot></table-wrap></p><p>Kf misincorporated both A and G opposite
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua. Compared to the normal incorporation of
dCTP opposite Gua, <italic toggle="yes">k</italic><sub>cat</sub> was considerably lower
in all cases
and <italic toggle="yes">K</italic><sub>m</sub> was much higher. With the
HO-ethanoGua-containing template, a higher misincorporation frequency was seen
for dATP than for dGTP. With the
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua-containing
template, the opposite pattern was observed. However,
the
misincorporation frequencies cannot be considered absolute
because the C incorporation is considered to involve a
slipped
frameshift intermediate (<italic toggle="yes">vide
</italic><italic toggle="yes">supra</italic>).
</p><p>With T7, <italic toggle="yes">k</italic><sub>cat</sub> values for all
incorporations opposite
modified Gs were considerably lower than those for dCTP
opposite G, but the <italic toggle="yes">K</italic><sub>m</sub> values were not altered.
Little
misincorporation was seen with the 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
template. The
misincorporation frequency measured with the HO-ethanoGua template indicated that the enzyme inserted dATP as
readily as dCTP.
</p></sec><sec id="d7e2112"><title>Discussion</title><p>Procedures were developed for the incorporation of
the
DNA adducts 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua into
defined
oligonucleotides, which were purified, characterized, and
used in <italic toggle="yes">in vitro</italic> misincorporation studies. The results
are of
interest in terms of understanding the potential genotoxic
properties of these two adducts and their possible
contributions in tumors caused by agents that yield these adducts.
</p><p>Both 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua
strongly blocked
replication with all polymerases examined (Figures <xref rid="bi962526vf00004"></xref>−8).
This appears to be the result of a decreased rate of
incorporation at the position opposite the modified base
in
most cases (Figures <xref rid="bi962526vf00003"></xref>−7), although more detailed studies
are in order to determine which step(s) in the
incorporation
is perturbed. With Kf, a strong block seems to occur
after
one base is incorporated. The purines A and G were
readily
incorporated opposite both 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua
by several polymerases (Tables <xref rid="bi962526vt00002"></xref> and <xref rid="bi962526vt00004"></xref>). We also
found
that pol I, pol II, and T7 are able to extend the primer
after
−1 or −2 base slippages at the adduct site (Figure <xref rid="bi962526vf00008"></xref>D
and
Table <xref rid="bi962526vt00002"></xref>), and we detected some 17- and 18-mer bands in
addition to the full-length extension product.
</p><p>Some differences between the effects of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua were noted. dATP was inserted more
readily
opposite HO-ethanoGua with several of the polymerases.
This result is of interest in that the level of
HO-ethanoGua
formed by treatment of DNA with 2-chlorooxirane is more
than 1 order of magnitude higher than that of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
(Müller et al., 1997). However, these results will need
to
be considered in the context of further work on rates of
DNA
repair and mutagenesis in cellular systems. The
steady-state
kinetic studies indicate a greater tendency for misincorporation opposite HO-ethanoGua than 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
with T7
(Table <xref rid="bi962526vt00004"></xref>).<xref rid="atyp_ref3" ref-type="bibr"></xref> It is not possible to make a
quantitative
interpretation of the Kf misincorporation frequencies
(Table
<xref rid="bi962526vt00004"></xref>) because of the apparent tendency of the template to
slip
rather than incorporate at the site of the adduct (Scheme
<xref rid="bi962526vh00004"></xref>
and Figure <xref rid="bi962526vf00008"></xref>B).
<fig id="bi962526vh00004" position="float" fig-type="scheme" orientation="portrait"><label>4</label><caption><p>Postulated Template Slippage in Kf Misincorporation Events</p></caption><graphic xlink:href="bi962526vh00004.gif" position="float" orientation="portrait"></graphic></fig></p><p>We noted some similarities in comparing our results
with
those described recently by Hashim and Marnett (1996) on
the effect of propanoG on the fidelity of Kf in the same
sequence context. PropanoG and the derivatives shown
in
Scheme <xref rid="bi962526vh00005"></xref> differ from
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua in
the
size of the exocyclic ring. Hashim and Marnett (1996)
reported primarily misincorporation of dATP opposite propanoG in the full-length extended product with Kf.
Interestingly, we found that such miscoding is also detectable
using
pol II, T7, and pol β opposite HO-ethanoGua; with
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, the same misincorporation is seen with pol II but not
with T7 or pol β. As in the case of propanoG and Kf
(Hashim & Marnett, 1996), the most common frame shift
mutation corresponded to a one-base deletion at the adduct
site with all of the enzymes studied (except with pol β,
where
only full-length extended product was detectable). With
Kf
in particular, we were able to detect substantial two-base
deletion opposite 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and HO-ethanoGua,
to a greater
extent than that detected opposite propanoG under the same
conditions (Hashim & Marnett, 1996). The one-base
deletion
opposite propanoG was also explained in previous work
(Shibutani & Grollman, 1993) by the slippage between A at
the 3‘-primer terminus and the T residue 5‘ to the adduct
site and can be extrapolated to our two adducts. In
contrast,
the two-base slippage (resulting in a two-base deletion at
the adduct site) cannot be explained in our case by C
incorporation opposite the adducts, since there was a
negligible amount of C incorporation opposite
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
and no C incorporation opposite HO-ethanoGua.
However,
this two-base deletion was more pronounced in the template
containing 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua than HO-ethanoGua, when
extensions
were performed with Kf under the same conditions. The
similar structures of M<sub>1</sub>G, the unsaturated form of
propanoG
[conjugation product of malondialdehyde (Seto et al.,
1983;
Basu et al., 1988), Scheme <xref rid="bi962526vh00005"></xref>], and 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua
should provide
an interesting comparison of misincorporation, slippage,
and
extension by several polymerases and are the focus of
further
investigations.
<fig id="bi962526vh00005" position="float" fig-type="scheme" orientation="portrait"><label>5</label><caption><p>Structures of Related Six-Membered Exocyclic Ring Gua DNA Adducts</p></caption><graphic xlink:href="bi962526vh00005.gif" position="float" orientation="portrait"></graphic></fig></p><p>Incorporation of T opposite the Gua adducts was
observed
only in the full-length product from the primer extension
with Kf and the HO-ethanoGua-containing template (Table
<xref rid="bi962526vt00002"></xref>). The corresponding mutation was seen in an <italic toggle="yes">in
vivo</italic>
mutagenesis study with this adduct, along with mutations
corresponding to incorporation of G and A.<xref rid="atyp_ref4" ref-type="bibr"></xref>
However, a
high level of mutations corresponding to insertion of T
opposite 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua was seen in the study,
regardless of
whether <italic toggle="yes">uvrA</italic><sup>+</sup> or
<italic toggle="yes">uvrA</italic><sup>-</sup> <italic toggle="yes">E. coli</italic> cells were used.
This
misincorporation, dominant in the bacterial study, was not
seen with any of the polymerases examined here, except at
a minor level with pol II (Table <xref rid="bi962526vt00002"></xref>). Thus, it appears
that
both of the adducts studied here are able to direct the
incorporation of <italic toggle="yes">all four</italic> dNTPs opposite the site (we
assume
that pol III is the polymerase producing the mutations
<italic toggle="yes">in
vivo</italic>). This pairing with all of the four dNTPs is not
simply
the result of a lack of information, since the different
polymerases all have their own preferences. In the case
of
1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua, the adduct is relatively stable,
unless an enamine
tautomer exists (Guengerich et al., 1993). With HO-ethanoGua, the possibility that different forms of the
adduct
may be favored with individual polymerases (and also
different sequence contexts) and favor different misincorporations exists, i.e., the ring-opened form
<italic toggle="yes">N</italic><sup>2</sup>-(2-oxoethyl)Gua, its hydrate [CH(OH)<sub>2</sub>], and the two isomers of
the
cyclic hemiaminal HO-ethanoGua (Scheme <xref rid="bi962526vh00001"></xref>).
Nevertheless,
the case can be made that only very limited insight into
mechanisms of mispairing can be obtained from studies with
oligomers in the absence of polymerases.
</p><p>In conclusion, 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua are both
capable of blocking polymerization and miscoding when
examined in defined oligomers. With several
polymerases,
there is a tendency to incorporate A and G opposite both
of
these adducts. Although the closed form of
HO-ethanoGua
differs from 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua only in the elements
of H<sub>2</sub>O, the
former tends to be more likely to direct the
misincorporation
of A and to produce a ladder of products extended by one
base at a time (Figures <xref rid="bi962526vf00003"></xref>−6). This phenomenon may
be
the result of an increased <italic toggle="yes">k</italic><sub>off</sub> rate for the
oligomer−polymerase complex, i.e., more distributive mechanism.
Efforts to define alterations in rates of individual steps
due
to the presence of adducts are in progress. Another
major
conclusion from this work is that a single adduct can
produce
quite different mutations, not only quantitatively but
also
qualitatively, depending upon the
polymerase. Previous
studies on 8-oxo-7,8-dihydroGua have revealed differences
among various polymerases in the extent to which A is
inserted instead of C (Shibutani et al., 1991; Lowe &
Guengerich, 1996; Furge & Guengerich, 1997), although
insertion of G and T has not been reported. Very
recently,
Shibutani et al. (1996) reported the misincorporation of
different bases at another ε-modified lesion,
3,<italic toggle="yes">N</italic><sup>4</sup>-ε-cytosine,
by individual polymerases.
</p><p>In summary, both 1,<italic toggle="yes">N</italic><sup>2</sup>-ε-Gua and
HO-ethanoGua have
been found to have miscoding potential and should be
included in considerations of the genotoxicity of the set
of
DNA adducts derived from vinyl halides and other carcinogens that are activated to similar epoxides or equivalent
alkylating species.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank B. Nobes and Drs. A. Chaudhary and F. J.
Belas
for mass spectral analysis, P. Horton for oligonucleotide
synthesis, Dr. L. Sowers for providing <italic toggle="yes">L. helveticus</italic><italic toggle="yes">trans</italic>-<italic toggle="yes">N</italic>-deoxyribosylase preparations used in the early studies,
Drs.
D. Tsarouhtsis and M. Hashim for technical suggestions, L.
L. Furge for preparing four of the polymerases used in the
study and for comments on the manuscript, and E. Rochelle
for assistance in preparation of the manuscript.
</p></ack><notes notes-type="si"><sec id="d7e2310"><title><ext-link xlink:href="/doi/suppl/10.1021%2Fbi962526v">Supporting Information Available</ext-link></title><p>CD spectra of HO-ethanoGua derivatives, a
polyacrylamide gel electrophoretogram of the HO-ethanoGua-containing 19-mer, and ES mass spectra of oligonucleotides (5
pages). Ordering information is given on any current
masthead page.
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The <italic toggle="yes">K</italic><sub>m</sub> value found (for dCTP) for the unmodified oligonucleotide and Kf is the same as that reported by Hashim and Marnett (1996); a large increase was seen with all incorporated dNTPs, as also observed here. The physical meaning of <italic toggle="yes">K</italic><sub>m</sub> in polymerase assays is not clear (Johnson, 1993); <italic toggle="yes"> K</italic><sub>m</sub> is certainly not a simple dissocation constant. <italic toggle="yes">K</italic><sub>d</sub> values must be obtained through more complex pre-steady-state experiments (Johnson, 1993; Lowe & Guengerich, 1996; Furge & Guengerich, 1997).</mixed-citation></ref><ref id="bi962526vb00004"><mixed-citation><comment>S. Langouët, S. P. Fink, M. Müller, L. J. Marnett, and F. P. Guengerich, unpublished results.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†</title></titleInfo><titleInfo contentType="CDATA"><title>Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†</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><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: Abt. Arbeits-und Sozialmedizinder Georg-August-Universität Göttingen, Waldweg 37, D-37073Göttingen,Germany.</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. 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">1997-05-20</dateCreated><dateIssued encoding="w3cdtf">1997-05-20</dateIssued><copyrightDate encoding="w3cdtf">1997</copyrightDate></originInfo><note type="footnote" ID="bi962526vAF2"> This research was supported in part by United States Public Health Service Grants R35 CA44353 and P30 ES00267. M.M. was supported in part by a fellowship from the Deutsche Forschungsgemeinschaft.</note><note type="footnote" ID="bi962526vAF7"> Abstract published in Advance ACS Abstracts, May 1, 1997.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>1,N2-Ethenoguanine (1,N2-ε-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5‘-CAGTGGGTG*TCCGAATTGA-3‘, were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H218O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3‘-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-ε-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, −1 and −2 base frame shifts were detected with both 1,N2-ε-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in kcat for all dNTP incorporations compared to the normal G·dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-ε-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.</abstract><note type="footnote" ID="bi962526vAF2"> This research was supported in part by United States Public Health Service Grants R35 CA44353 and P30 ES00267. 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(1987) Carcinogenesis 8, 745−747.</note></relatedItem><relatedItem type="references" ID="bi962526vb00001" displayLabel="bibbi962526vb00001"><titleInfo><title>Abbreviations: ε, etheno; HO-ethanoGua, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine; HO-ethanodGuo, HO-ethanoGua deoxyribose; AcO-ethanoGua, 5,6,7,9-tetrahydro-7-acetoxy-9-oxoimidazo[1,2-a]purine; Tris, tris(hydroxymethyl)aminomethane; MOPS, 3-(N-morpholino)propanesulfonic acid; EDTA, (ethylenedinitrilo)tetraacetic acid; Gua, guanine; Cyt, cytosine; Ade, adenine; dGuo, deoxyguanosine; dThd, thymidine; dCyd, deoxycytidine; dAdo, deoxyadenosine; propanoG, 1,N2-propanoguanine; M1G, pyrimido[1,2-a]purin-10(3H)-one; HPLC, high-performance liquid chromatography; TLC, thin layer chromatography; CGE, capillary gel electrophoresis; MS, mass spectrometry; ES, electrospray; FAB, fast atom bombardment; Kf, Klenow fragment (of pol I) exo-; RT, human immunodeficiency virus-1 reverse transcriptase; pol II, polymerase II exo-; T7, polymerase T7 exo-/thioredoxin mixture; pol β, rat polymerase β.</title></titleInfo><note type="content-in-line">Abbreviations: ε, etheno; HO-ethanoGua, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine; HO-ethanodGuo, HO-ethanoGua deoxyribose; AcO-ethanoGua, 5,6,7,9-tetrahydro-7-acetoxy-9-oxoimidazo[1,2-a]purine; Tris, tris(hydroxymethyl)aminomethane; MOPS, 3-(N-morpholino)propanesulfonic acid; EDTA, (ethylenedinitrilo)tetraacetic acid; Gua, guanine; Cyt, cytosine; Ade, adenine; dGuo, deoxyguanosine; dThd, thymidine; dCyd, deoxycytidine; dAdo, deoxyadenosine; propanoG, 1,N2-propanoguanine; M1G, pyrimido[1,2-a]purin-10(3H)-one; HPLC, high-performance liquid chromatography; TLC, thin layer chromatography; CGE, capillary gel electrophoresis; MS, mass spectrometry; ES, electrospray; FAB, fast atom bombardment; Kf, Klenow fragment (of pol I) exo-; RT, human immunodeficiency virus-1 reverse transcriptase; pol II, polymerase II exo-; T7, polymerase T7 exo-/thioredoxin mixture; pol β, rat polymerase β.</note></relatedItem><relatedItem type="references" ID="bi962526vb00002" displayLabel="bibbi962526vb00002"><titleInfo><title>The strategy of inserting a 2-fluoroinosine group into the oligomer, adding 2-aminoacetaldehyde dimethyl acetal, and then closing the ring (Decorte et al., 1996; Guengerich et al., 1993) was considered but not attempted because studies with the nucleoside indicated that the glycosidic bond was hydrolyzed under all conditions used to cleave the acetal.</title></titleInfo><note type="content-in-line">The strategy of inserting a 2-fluoroinosine group into the oligomer, adding 2-aminoacetaldehyde dimethyl acetal, and then closing the ring (Decorte et al., 1996; Guengerich et al., 1993) was considered but not attempted because studies with the nucleoside indicated that the glycosidic bond was hydrolyzed under all conditions used to cleave the acetal.</note></relatedItem><relatedItem type="references" ID="bi962526vb00003" displayLabel="bibbi962526vb00003"><titleInfo><title>K</title></titleInfo><note type="content-in-line">Some comment is in order about the Km values in Table 4. The Km value found (for dCTP) for the unmodified oligonucleotide and Kf is the same as that reported by Hashim and Marnett (1996); a large increase was seen with all incorporated dNTPs, as also observed here. The physical meaning of Km in polymerase assays is not clear (Johnson, 1993); Km is certainly not a simple dissocation constant. Kd values must be obtained through more complex pre-steady-state experiments (Johnson, 1993; Lowe & Guengerich, 1996; Furge & Guengerich, 1997).</note></relatedItem><relatedItem type="references" ID="bi962526vb00004" displayLabel="bibbi962526vb00004"><titleInfo><title>S. Langouët, S. P. Fink, M. Müller, L. J. Marnett, and F. P. Guengerich, unpublished results.</title></titleInfo><note type="content-in-line">S. Langouët, S. P. Fink, M. Müller, L. J. Marnett, and F. P. Guengerich, unpublished results.</note></relatedItem><identifier type="istex">2010D51B69180BED3FDB60A600FBAF86BADAEA41</identifier><identifier type="ark">ark:/67375/TPS-69F58Q91-C</identifier><identifier type="DOI">10.1021/bi962526v</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 1997 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-69F58Q91-C/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-69F58Q91-C/annexes.gif</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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|texte= Misincorporation of dNTPs Opposite 1,N2-Ethenoguanine and 5,6,7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in Oligonucleotides by Escherichia coli Polymerases I exo- and II exo-, T7 Polymerase exo-, Human Immunodeficiency Virus-1 Reverse Transcriptase, and Rat Polymerase β†
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