Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†
Identifieur interne : 001A83 ( Istex/Corpus ); précédent : 001A82; suivant : 001A84Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†
Auteurs : Juan P. Guaitiao ; Roberto A. Zú Iga ; Monica J. Roth ; Oscar LeonSource :
- Biochemistry [ 0006-2960 ] ; 2004.
Abstract
An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.
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DOI: 10.1021/bi035454y
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Isolated RNase H Domain of HIV-1 Reverse Transcriptase<ref type="bib" target="#bi035454yAF2">†</ref></title><author><name sortKey="Guaitiao, Juan P" sort="Guaitiao, Juan P" uniqKey="Guaitiao J" first="Juan P." last="Guaitiao">Juan P. Guaitiao</name><affiliation><mods:affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</mods:affiliation></affiliation><affiliation><mods:affiliation> Universidad de Chile.</mods:affiliation></affiliation></author><author><name sortKey="Zu Iga, Roberto A" sort="Zu Iga, Roberto A" uniqKey="Zu Iga R" first="Roberto A." last="Zú Iga">Roberto A. Zú Iga</name><affiliation><mods:affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</mods:affiliation></affiliation><affiliation><mods:affiliation> Universidad de Chile.</mods:affiliation></affiliation></author><author><name sortKey="Roth, Monica J" sort="Roth, Monica J" uniqKey="Roth M" first="Monica J." last="Roth">Monica J. Roth</name><affiliation><mods:affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Medicine and Dentistry of New Jersey−Robert WoodJohnson Medical School.</mods:affiliation></affiliation></author><author><name sortKey="Leon, Oscar" sort="Leon, Oscar" uniqKey="Leon O" first="Oscar" last="Leon">Oscar Leon</name><affiliation><mods:affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</mods:affiliation></affiliation><affiliation><mods:affiliation> Universidad de Chile.</mods:affiliation></affiliation><affiliation><mods:affiliation> Corresponding author. Tel: 56 63 2 678 6858. Fax 56 63 2 6786124. E-mail: oleon@med.uchile.cl.</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">2004</date><date type="published">2004</date><biblScope unit="vol">43</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="1302">1302</biblScope><biblScope unit="page" to="1308">1308</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">An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>rnase</json:string><json:string>trna</json:string><json:string>c2dt</json:string><json:string>hybrid</json:string><json:string>biochemistry</json:string><json:string>primer</json:string><json:string>lysine</json:string><json:string>unmodified substrate</json:string><json:string>thrombin</json:string><json:string>biol</json:string><json:string>viral</json:string><json:string>cleavage</json:string><json:string>crosslinking</json:string><json:string>oligonucleotide</json:string><json:string>peptide</json:string><json:string>covalent</json:string><json:string>base c2dt</json:string><json:string>trypsin</json:string><json:string>amino</json:string><json:string>unmodified</json:string><json:string>amino group</json:string><json:string>covalent complex</json:string><json:string>scissile bond</json:string><json:string>urea</json:string><json:string>major groove</json:string><json:string>trna removal</json:string><json:string>amino acid</json:string><json:string>trna primer</json:string><json:string>nucleic</json:string><json:string>ethanol</json:string><json:string>thrombin digestion</json:string><json:string>rnase acid complex</json:string><json:string>time course analysis</json:string><json:string>nitrocellulose filter</json:string></teeft></keywords><author><json:item><name>GUAITIAO Juan P.</name><affiliations><json:string>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</json:string><json:string>Universidad de Chile.</json:string></affiliations></json:item><json:item><name>ZúñIGA Roberto A.</name><affiliations><json:string>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</json:string><json:string>Universidad de Chile.</json:string></affiliations></json:item><json:item><name>ROTH Monica J.</name><affiliations><json:string>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</json:string><json:string>University of Medicine and Dentistry of New Jersey−Robert WoodJohnson Medical School.</json:string></affiliations></json:item><json:item><name>LEON Oscar</name><affiliations><json:string>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</json:string><json:string>Universidad de Chile.</json:string><json:string>Corresponding author. Tel: 56 63 2 678 6858. Fax 56 63 2 6786124. E-mail: oleon@med.uchile.cl.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-361D4HN4-Z</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.</abstract><qualityIndicators><score>9.595</score><pdfWordCount>4967</pdfWordCount><pdfCharCount>28829</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>7</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>710</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>false</refBibsNative><abstractWordCount>219</abstractWordCount><abstractCharCount>1479</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†</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>43</volume><issue>5</issue><pages><first>1302</first><last>1308</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-361D4HN4-Z</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2004</publicationDate><copyrightDate>2004</copyrightDate><doi><json:string>10.1021/bi035454y</json:string></doi><id>9C8C171749D38E672C7D069ED3336F5E383E9590</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-361D4HN4-Z/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-361D4HN4-Z/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-361D4HN4-Z/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-361D4HN4-Z/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the
Isolated RNase H Domain of HIV-1 Reverse Transcriptase<ref type="bib" target="#bi035454yAF2">†</ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2004 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2004</date><date type="Copyright" when="2004">2004</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">Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the
Isolated RNase H Domain of HIV-1 Reverse Transcriptase<ref type="bib" target="#bi035454yAF2">†</ref></title><author xml:id="author-0000"><persName><surname>Guaitiao</surname><forename type="first">Juan P.</forename></persName><affiliation><orgName type="group">Programa de Virología</orgName><orgName type="institution">Instituto de Ciencias Biomédicas</orgName><orgName type="department">Facultad de Medicina</orgName><orgName type="institution">Universidad de Chile</orgName><address><addrLine>Independencia 1027</addrLine><addrLine>Santiago</addrLine><country key="CL" xml:lang="en">CHILE</country><addrLine>and Department of Biochemistry</addrLine><addrLine>University of Medicine and Dentistry of New Jersey−Robert Wood Johnson Medical School</addrLine><addrLine>675 Hoes Lane</addrLine><addrLine>Piscataway</addrLine><addrLine>New Jersey 08854 USA</addrLine></address></affiliation><note place="foot" n="bi035454yAF3"><ref>‡</ref><p>
Universidad de Chile.</p></note></author><author xml:id="author-0001"><persName><surname>Zúñiga</surname><forename type="first">Roberto A.</forename></persName><affiliation><orgName type="group">Programa de Virología</orgName><orgName type="institution">Instituto de Ciencias Biomédicas</orgName><orgName type="department">Facultad de Medicina</orgName><orgName type="institution">Universidad de Chile</orgName><address><addrLine>Independencia 1027</addrLine><addrLine>Santiago</addrLine><country key="CL" xml:lang="en">CHILE</country><addrLine>and Department of Biochemistry</addrLine><addrLine>University of Medicine and Dentistry of New Jersey−Robert Wood Johnson Medical School</addrLine><addrLine>675 Hoes Lane</addrLine><addrLine>Piscataway</addrLine><addrLine>New Jersey 08854 USA</addrLine></address></affiliation><note place="foot" n="bi035454yAF3"><ref>‡</ref><p>
Universidad de Chile.</p></note></author><author xml:id="author-0002"><persName><surname>Roth</surname><forename type="first">Monica J.</forename></persName><affiliation><orgName type="group">Programa de Virología</orgName><orgName type="institution">Instituto de Ciencias Biomédicas</orgName><orgName type="department">Facultad de Medicina</orgName><orgName type="institution">Universidad de Chile</orgName><address><addrLine>Independencia 1027</addrLine><addrLine>Santiago</addrLine><country key="CL" xml:lang="en">CHILE</country><addrLine>and Department of Biochemistry</addrLine><addrLine>University of Medicine and Dentistry of New Jersey−Robert Wood Johnson Medical School</addrLine><addrLine>675 Hoes Lane</addrLine><addrLine>Piscataway</addrLine><addrLine>New Jersey 08854 USA</addrLine></address></affiliation><note place="foot" n="bi035454yAF4"><ref>§</ref><p>
University of Medicine and Dentistry of New Jersey−Robert Wood
Johnson Medical School.</p></note></author><author xml:id="author-0003" role="corresp"><persName><surname>Leon</surname><forename type="first">Oscar</forename></persName><note place="foot" n="bi035454yAF3"><ref>‡</ref><p>
Universidad de Chile.</p></note><affiliation role="corresp"> Corresponding author. Tel: 56 63 2 678 6858. Fax 56 63 2 678 6124. E-mail: oleon@med.uchile.cl.</affiliation></author><idno type="istex">9C8C171749D38E672C7D069ED3336F5E383E9590</idno><idno type="ark">ark:/67375/TPS-361D4HN4-Z</idno><idno type="DOI">10.1021/bi035454y</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">2004</date><date type="published">2004</date><biblScope unit="vol">43</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="1302">1302</biblScope><biblScope unit="page" to="1308">1308</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>An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically
removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity
are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition
of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed
toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the
DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of
Mn<hi rend="superscript">2+</hi>. The Mn<hi rend="superscript">2+</hi> titration of cross-linking paralleled the Mn<hi rend="superscript">2+</hi> requirement for activity. Modified substrate
quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the
cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex
revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating
that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of
K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity.
On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the
tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant
polypurine tract (PPT) substrate within the HIV-1 RT.
</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-2.0.3"><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/bi035454y</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the
Isolated RNase H Domain of HIV-1 Reverse Transcriptase<xref rid="bi035454yAF2">†</xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Guaitiao</surname><given-names>Juan P.</given-names></name><xref rid="bi035454yAF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Zúñiga</surname><given-names>Roberto A.</given-names></name><xref rid="bi035454yAF3">‡</xref></contrib><contrib contrib-type="author"><name><surname>Roth</surname><given-names>Monica J.</given-names></name><xref rid="bi035454yAF4">§</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Leon</surname><given-names>Oscar</given-names></name><xref rid="bi035454yAF1">*</xref><xref rid="bi035454yAF3">‡</xref></contrib><aff>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,
Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry of
New Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA
</aff></contrib-group><author-notes><fn id="bi035454yAF3"><label>‡</label><p>
Universidad de Chile.</p></fn><fn id="bi035454yAF4"><label>§</label><p>
University of Medicine and Dentistry of New Jersey−Robert Wood
Johnson Medical School.</p></fn><corresp id="bi035454yAF1">
Corresponding author. Tel: 56 63 2 678 6858. Fax 56 63 2 678
6124. E-mail: oleon@med.uchile.cl.</corresp></author-notes><pub-date pub-type="epub"><day>13</day><month>01</month><year>2004</year></pub-date><pub-date pub-type="ppub"><day>10</day><month>02</month><year>2004</year></pub-date><volume>43</volume><issue>5</issue><fpage>1302</fpage><lpage>1308</lpage><history><date date-type="received"><day>14</day><month>08</month><year>2003</year></date><date date-type="rev-recd"><day>21</day><month>11</month><year>2003</year></date><date date-type="asap"><day>13</day><month>01</month><year>2004</year></date><date date-type="issue-pub"><day>10</day><month>02</month><year>2004</year></date></history><permissions><copyright-statement>Copyright © 2004 American Chemical Society</copyright-statement><copyright-year>2004</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically
removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity
are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition
of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed
toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the
DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of
Mn<sup>2+</sup>. The Mn<sup>2+</sup> titration of cross-linking paralleled the Mn<sup>2+</sup> requirement for activity. Modified substrate
quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the
cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex
revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating
that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of
K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity.
On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the
tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant
polypurine tract (PPT) substrate within the HIV-1 RT.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi035454y</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi035454yAF2"><label>†</label><p>
This work was supported by Grants RO1 CA90174 Award and
Fondecyt 198982.</p></notes></front><body></body><back></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†</title></titleInfo><titleInfo contentType="CDATA"><title>Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†</title></titleInfo><name type="personal"><namePart type="family">GUAITIAO</namePart><namePart type="given">Juan P.</namePart><affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</affiliation><affiliation> Universidad de Chile.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">ZúñIGA</namePart><namePart type="given">Roberto A.</namePart><affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</affiliation><affiliation> Universidad de Chile.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">ROTH</namePart><namePart type="given">Monica J.</namePart><affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</affiliation><affiliation> University of Medicine and Dentistry of New Jersey−Robert WoodJohnson Medical School.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">LEON</namePart><namePart type="given">Oscar</namePart><affiliation>Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile,Independencia 1027, Santiago, Chile, and Department of Biochemistry, University of Medicine and Dentistry ofNew Jersey−Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854 USA</affiliation><affiliation> Universidad de Chile.</affiliation><affiliation> Corresponding author. Tel: 56 63 2 678 6858. Fax 56 63 2 6786124. E-mail: oleon@med.uchile.cl.</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">2004-01-13</dateCreated><dateIssued encoding="w3cdtf">2004-02-10</dateIssued><copyrightDate encoding="w3cdtf">2004</copyrightDate></originInfo><note type="footnote" ID="bi035454yAF2"> This work was supported by Grants RO1 CA90174 Award and Fondecyt 198982.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.</abstract><note type="footnote" ID="bi035454yAF2"> This work was supported by Grants RO1 CA90174 Award and Fondecyt 198982.</note><relatedItem type="host"><titleInfo><title>Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>Biochemistry</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0006-2960</identifier><identifier type="eISSN">1520-4995</identifier><identifier type="acspubs">bi</identifier><identifier type="coden">BICHAW</identifier><identifier type="uri">pubs.acs.org/biochemistry</identifier><part><date>2004</date><detail type="volume"><caption>vol.</caption><number>43</number></detail><detail type="issue"><caption>no.</caption><number>5</number></detail><extent unit="pages"><start>1302</start><end>1308</end></extent></part></relatedItem><identifier type="istex">9C8C171749D38E672C7D069ED3336F5E383E9590</identifier><identifier type="ark">ark:/67375/TPS-361D4HN4-Z</identifier><identifier type="DOI">10.1021/bi035454y</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2004 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-361D4HN4-Z/record.json</uri></json:item></metadata><annexes><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/9C8C171749D38E672C7D069ED3336F5E383E9590/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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