DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase
Identifieur interne : 001D99 ( Istex/Corpus ); précédent : 001D98; suivant : 001E00DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase
Auteurs : Young Hwang ; Minkyu Park ; Wolfgang H. Fischer ; Alex Burgin Jr. ; Frederic BushmanSource :
- Virology [ 0042-6822 ] ; 1999.
English descriptors
- Teeft :
- Academic press, Active site tyrosine, Additional monomers, Aggregated complexes, Amino, Amino acid sequencing, Amino acids, Apparent rate, Assay, Base pair, Binding activity, Catalytic, Catalytic domain, Catalytic domains, Cheng, Cleavage, Coding region, Complex formation, Contagiosum, Control reactions, Correct contacts, Covalent, Covalent catalysis, Covalent complexes, Covalent form, Digestion, Digestion products, Domain, Domain structure, Edman degradation, Enzyme, Escherichia coli, Expression vector, Fragment, Fragment encoding, High concentrations, Human topoisomerase, Hwang, Individual tubes, Initial binding, Initial cleavage step, Input substrate, Lambda integrase, Lambda integrase family, Linker, Linker region, Modest effects, Molluscum, Molluscum contagiosum, Molluscum contagiosum virus topoisomerase, Nacl, Optimal conditions, Other members, Partial proteolysis, Pentamer, Petersen, Phosphotyrosine linkage, Poxvirus, Poxvirus topoisomerase, Previous studies, Protein domains, Reaction mixture, Reaction mixtures, Reaction products, Recognition site, Relative noncovalent binding nucleotide sequence, Relaxation activity, Religation, Religation activity, Religation reaction, Scissile phosphate, Sequencing type, Short duplex extension, Shuman, Stable domains, Stable form, Structural studies, Suicide substrate, Sulfur atom, Topoisomerase, Trypsin, Unpublished data, Vaccinia, Vaccinia topoisomerase, Vaccinia virus, Various times, Wild type.
Abstract
Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.
Url:
DOI: 10.1006/viro.1999.9920
Links to Exploration step
ISTEX:5141E7E4B6937BFC2CA54509E78AA9F81271CB73Le document en format XML
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Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. 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Fischer</name><affiliations><json:string>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</json:string></affiliations></json:item><json:item><name>Alex Burgin, Jr.</name><affiliations><json:string>Biology Department, San Diego State University, San Diego, California, 92182-4614</json:string></affiliations></json:item><json:item><name>Frederic Bushman</name><affiliations><json:string>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-5S06TSB6-5</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.</abstract><qualityIndicators><score>9.664</score><pdfWordCount>5892</pdfWordCount><pdfCharCount>37927</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>533 x 723 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>222</abstractWordCount><abstractCharCount>1569</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</title><pmid><json:string>10502526</json:string></pmid><pii><json:string>S0042-6822(99)99920-8</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Virology</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0042-6822</json:string></issn><pii><json:string>S0042-6822(00)X0062-1</json:string></pii><volume>262</volume><issue>2</issue><pages><first>479</first><last>491</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1999</json:string></date><geogName></geogName><orgName><json:string>AIDS Research Program</json:string><json:string>University of California Universitywide</json:string><json:string>Biology Department, San Diego State University, San Diego, California</json:string><json:string>Grant R97</json:string><json:string>Grant AI46222</json:string></orgName><orgName_funder><json:string>Grant R97</json:string><json:string>Grant AI46222</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Studies</json:string><json:string>Allison Bocksruker</json:string><json:string>A. Burgin</json:string><json:string>La Jolla</json:string><json:string>Frederic Bushman</json:string></persName><placeName><json:string>Mannheim</json:string><json:string>America</json:string></placeName><ref_url><json:string>http://www.idealibrary.com</json:string></ref_url><ref_bibl><json:string>Sharma et al., 1994</json:string><json:string>Shuman et al., 1989</json:string><json:string>Massung et al., 1993</json:string><json:string>Hwang et al., 1999</json:string><json:string>Shuman, 1991</json:string><json:string>Petersen et al. (1997)</json:string><json:string>Hwang et al., 1999b</json:string><json:string>Shuman et al., 1988</json:string><json:string>Hwang et al., 1998</json:string><json:string>Klemperer et al., 1995</json:string><json:string>Schwartz and Myskowski, 1992</json:string><json:string>Petersen and Gerstoft, 1992</json:string><json:string>Hwang et al., 1999a, 1998</json:string><json:string>Porter et al., 1992</json:string><json:string>Hwang et al., 1999a</json:string><json:string>Petersen et al., 1997</json:string><json:string>Wang, 1996</json:string><json:string>Kwon et al., 1997</json:string><json:string>Fischer et al., 1991</json:string><json:string>Cheng et al., 1998</json:string><json:string>Sekiguchi and Shuman, 1996</json:string><json:string>Gottlieb and Myskowski, 1994</json:string><json:string>Sambrook et al., 1989</json:string><json:string>Redinbo et al., 1998</json:string><json:string>Stewart et al., 1998</json:string><json:string>Senkevich et al., 1996</json:string><json:string>Cheng and Shuman, 1998</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-5S06TSB6-5</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - virology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - virology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Virology</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>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1006/viro.1999.9920</json:string></doi><id>5141E7E4B6937BFC2CA54509E78AA9F81271CB73</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-5S06TSB6-5/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-5S06TSB6-5/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-5S06TSB6-5/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1999 Academic Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1999</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Regular Article</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</title><author xml:id="author-0000"><persName><forename type="first">Young</forename><surname>Hwang</surname></persName><affiliation>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Minkyu</forename><surname>Park</surname></persName><affiliation>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Wolfgang H.</forename><surname>Fischer</surname></persName><affiliation>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Alex</forename><surname>Burgin, Jr.</surname></persName><affiliation>Biology Department, San Diego State University, San Diego, California, 92182-4614</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Frederic</forename><surname>Bushman</surname></persName><note type="biography">To whom reprint requests should be addressed at 10010 North Torrey Pines Road, La Jolla, CA 92037.</note><affiliation>To whom reprint requests should be addressed at 10010 North Torrey Pines Road, La Jolla, CA 92037.</affiliation><affiliation>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation></author><idno type="istex">5141E7E4B6937BFC2CA54509E78AA9F81271CB73</idno><idno type="ark">ark:/67375/6H6-5S06TSB6-5</idno><idno type="DOI">10.1006/viro.1999.9920</idno><idno type="PII">S0042-6822(99)99920-8</idno></analytic><monogr><title level="j">Virology</title><title level="j" type="abbrev">YVIRO</title><idno type="pISSN">0042-6822</idno><idno type="PII">S0042-6822(00)X0062-1</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">262</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="479">479</biblScope><biblScope unit="page" to="491">491</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.</p></abstract></profileDesc><revisionDesc><change when="1999-07-06">Modified</change><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-5S06TSB6-5/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YVIRO</jid><aid>99920</aid><ce:pii>S0042-6822(99)99920-8</ce:pii><ce:doi>10.1006/viro.1999.9920</ce:doi><ce:copyright type="full-transfer" year="1999">Academic Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Regular Article</ce:textfn></ce:dochead><ce:title>DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</ce:title><ce:author-group><ce:author><ce:given-name>Young</ce:given-name><ce:surname>Hwang</ce:surname><ce:cross-ref refid="A1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Minkyu</ce:given-name><ce:surname>Park</ce:surname><ce:cross-ref refid="A2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Wolfgang H.</ce:given-name><ce:surname>Fischer</ce:surname><ce:cross-ref refid="A2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Alex</ce:given-name><ce:surname>Burgin</ce:surname><ce:suffix>Jr.</ce:suffix><ce:cross-ref refid="A3"><ce:sup>c</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Frederic</ce:given-name><ce:surname>Bushman</ce:surname><ce:cross-ref refid="A1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:affiliation id="A1"><ce:label>a</ce:label><ce:textfn>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</ce:textfn></ce:affiliation><ce:affiliation id="A2"><ce:label>b</ce:label><ce:textfn>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</ce:textfn></ce:affiliation><ce:affiliation id="A3"><ce:label>c</ce:label><ce:textfn>Biology Department, San Diego State University, San Diego, California, 92182-4614</ce:textfn></ce:affiliation><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para>To whom reprint requests should be addressed at 10010 North Torrey Pines Road, La Jolla, CA 92037.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="15" month="6" year="1999"></ce:date-received><ce:date-revised day="6" month="7" year="1999"></ce:date-revised><ce:date-accepted day="20" month="7" year="1999"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in <ce:italic>Escherichia coli</ce:italic> and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids <ce:italic>in vitro,</ce:italic> albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase</title></titleInfo><name type="personal"><namePart type="given">Young</namePart><namePart type="family">Hwang</namePart><affiliation>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Minkyu</namePart><namePart type="family">Park</namePart><affiliation>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Wolfgang H.</namePart><namePart type="family">Fischer</namePart><affiliation>Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alex</namePart><namePart type="family">Burgin, Jr.</namePart><affiliation>Biology Department, San Diego State University, San Diego, California, 92182-4614</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Frederic</namePart><namePart type="family">Bushman</namePart><affiliation>Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037</affiliation><description>To whom reprint requests should be addressed at 10010 North Torrey Pines Road, La Jolla, CA 92037.</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><dateModified encoding="w3cdtf">1999-07-06</dateModified><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.</abstract><note type="content">Section title: Regular Article</note><relatedItem type="host"><titleInfo><title>Virology</title></titleInfo><titleInfo type="abbreviated"><title>YVIRO</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued></originInfo><identifier type="ISSN">0042-6822</identifier><identifier type="PII">S0042-6822(00)X0062-1</identifier><part><date>1999</date><detail type="volume"><number>262</number><caption>vol.</caption></detail><detail type="issue"><number>2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>265</start><end>493</end></extent><extent unit="pages"><start>479</start><end>491</end></extent></part></relatedItem><identifier type="istex">5141E7E4B6937BFC2CA54509E78AA9F81271CB73</identifier><identifier type="ark">ark:/67375/6H6-5S06TSB6-5</identifier><identifier type="DOI">10.1006/viro.1999.9920</identifier><identifier type="PII">S0042-6822(99)99920-8</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©1999</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-5S06TSB6-5/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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