Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system
Identifieur interne : 001C53 ( Istex/Corpus ); précédent : 001C52; suivant : 001C54Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system
Auteurs : Tomas Sinkunas ; Giedrius Gasiunas ; Christophe Fremaux ; Rodolphe Barrangou ; Philippe Horvath ; Virginijus SiksnysSource :
- The EMBO Journal [ 0261-4189 ] ; 2011-04-06.
Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA‐guided interference with virus proliferation. Cas3 contains N‐terminal HD phosphohydrolase and C‐terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single‐stranded DNA (ssDNA)‐stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP‐independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation‐of‐function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site‐directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.
Cas3 is an essential protein of unknown function required for CRISPR‐based bacteriophage immunity in bacteria. Here, the biochemical activities of Cas3 are demonstrated and mechanistic implications for immunity are discussed.
Url:
DOI: 10.1038/emboj.2011.41
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In Escherichia coli, Cas3 is essential for crRNA‐guided interference with virus proliferation. Cas3 contains N‐terminal HD phosphohydrolase and C‐terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single‐stranded DNA (ssDNA)‐stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP‐independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation‐of‐function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site‐directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.</div><div type="abstract">Cas3 is an essential protein of unknown function required for CRISPR‐based bacteriophage immunity in bacteria. 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et al, 2002</json:string><json:string>Hale et al, 2009</json:string><json:string>Matson, 1986</json:string><json:string>located in motif VI (Tanner and Linder, 2001</json:string><json:string>Biswas et al, 2009</json:string><json:string>located in Q-motif (Tanner et al, 2003</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/WNG-TCWFBFS5-V</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - cell biology</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 - developmental biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - General Immunology and Microbiology</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, 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<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract style="main"><p>Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In <hi rend="italic">Escherichia coli</hi>, Cas3 is essential for crRNA‐guided interference with virus proliferation. Cas3 contains N‐terminal HD phosphohydrolase and C‐terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and <hi rend="italic">in vitro</hi> functional analysis of the Cas3 protein of the <hi rend="italic">Streptococcus thermophilus</hi> CRISPR4 (Ecoli subtype) system. Cas3 possesses a single‐stranded DNA (ssDNA)‐stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP‐independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation‐of‐function mutants (ATPase<hi rend="superscript">+</hi>/nuclease<hi rend="superscript">−</hi> and ATPase<hi rend="superscript">−</hi>/nuclease<hi rend="superscript">+</hi>) were obtained by site‐directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.</p></abstract><abstract style="short"><p>Cas3 is an essential protein of unknown function required for CRISPR‐based bacteriophage immunity in bacteria. Here, the biochemical activities of Cas3 are demonstrated and mechanistic implications for immunity are discussed.</p></abstract><textClass><keywords><term xml:id="embj201141-kwd-0001">Cas3</term><term xml:id="embj201141-kwd-0002">CRISPR</term><term xml:id="embj201141-kwd-0003">Ecoli subtype</term><term xml:id="embj201141-kwd-0004">site‐directed mutagenesis</term><term xml:id="embj201141-kwd-0005"><hi rend="italic">Streptococcus thermophilus</hi></term></keywords><keywords rend="articleCategory"><term>Article</term></keywords><keywords rend="tocHeading1"><term>Article</term></keywords></textClass><textClass><keywords ana="subject"><term ref="http://psi.embo.org/14602075/EMBO36">RNA Biology</term><term ref="http://psi.embo.org/14602075/EMBO23">Microbiology, Virology & Host Pathogen Interaction</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2019-12-20" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-TCWFBFS5-V/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component type="serialArticle" version="2.0" xml:lang="en" xml:id="embj201141"><header><publicationMeta level="product"><publisherInfo><publisherName>John Wiley & Sons, Ltd</publisherName><publisherLoc>Chichester, UK</publisherLoc></publisherInfo><doi>10.1002/(ISSN)1460-2075</doi><issn type="print">0261-4189</issn><issn type="electronic">1460-2075</issn><idGroup><id type="product" value="EMBJ"></id></idGroup><titleGroup><title type="main" sort="THE EMBO JOURNAL">The EMBO Journal</title></titleGroup></publicationMeta><publicationMeta level="part" position="70"><doi>10.1002/emboj.v30.7</doi><copyright ownership="publisher">Copyright © 2013 Wiley Periodicals, Inc</copyright><numberingGroup><numbering type="journalVolume" number="30">30</numbering><numbering type="journalIssue">7</numbering></numberingGroup><coverDate startDate="2011-04-06">April 6, 2011</coverDate></publicationMeta><publicationMeta level="unit" position="170" type="article" status="forIssue"><doi>10.1038/emboj.2011.41</doi><idGroup><id type="unit" value="EMBJ201141"></id></idGroup><countGroup><count type="pageTotal" number="8"></count></countGroup><titleGroup>
<title type="articleCategory">Article</title><title type="tocHeading1">Article</title></titleGroup><copyright ownership="thirdParty">Copyright © 2011 European Molecular Biology Organization</copyright><eventGroup><event type="manuscriptReceived" date="2010-11-06"></event><event type="manuscriptAccepted" date="2011-02-01"></event><event type="publishedOnlineEarlyUnpaginated" date="2011-02-22"></event><event type="publishedOnlineFinalForm" date="2011-04-06"></event><event type="xmlConverted" agent="SPi Global Converter_TO_WileyML3Gv2.0 version:1.0; WileyML 3G Packaging Tool v1.0" date="2013-10-21"></event><event type="firstOnline" date="2011-02-22"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.1.7 mode:FullText,remove_FC" date="2014-11-04"></event></eventGroup><numberingGroup><numbering type="pageFirst">1335</numbering><numbering type="pageLast">1342</numbering></numberingGroup><correspondenceTo>Corresponding author. Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Tel.: +370 5 2602108; Fax: +370 5 2602116; E-mail: <email>siksnys@ibt.lt</email></correspondenceTo><subjectInfo><subject href="http://psi.embo.org/14602075/EMBO36">RNA Biology</subject><subject href="http://psi.embo.org/14602075/EMBO23">Microbiology, Virology & Host Pathogen Interaction</subject></subjectInfo><linkGroup><link type="toTypesetVersion" href="file:EMBJ.EMBJ201141.pdf"></link></linkGroup></publicationMeta><contentMeta><titleGroup><title type="main">Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system</title><title type="short">Cas3 nuclease/helicase</title></titleGroup><creators><creator creatorRole="author" xml:id="embj201141-cr-0001" affiliationRef="#embj201141-aff-0001"><personName><givenNames>Tomas</givenNames><familyName>Sinkunas</familyName></personName></creator><creator creatorRole="author" xml:id="embj201141-cr-0002" affiliationRef="#embj201141-aff-0001"><personName><givenNames>Giedrius</givenNames><familyName>Gasiunas</familyName></personName></creator><creator creatorRole="author" xml:id="embj201141-cr-0003" affiliationRef="#embj201141-aff-0002"><personName><givenNames>Christophe</givenNames><familyName>Fremaux</familyName></personName></creator><creator creatorRole="author" xml:id="embj201141-cr-0004" affiliationRef="#embj201141-aff-0003"><personName><givenNames>Rodolphe</givenNames><familyName>Barrangou</familyName></personName></creator><creator creatorRole="author" xml:id="embj201141-cr-0005" affiliationRef="#embj201141-aff-0002"><personName><givenNames>Philippe</givenNames><familyName>Horvath</familyName></personName></creator><creator creatorRole="author" xml:id="embj201141-cr-0006" affiliationRef="#embj201141-aff-0001" corresponding="yes"><personName><givenNames>Virginijus</givenNames><familyName>Siksnys</familyName></personName></creator></creators><affiliationGroup><affiliation countryCode="LT" type="organization" xml:id="embj201141-aff-0001"><orgName>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University</orgName><address><city>Vilnius</city><country>Lithuania</country></address></affiliation><affiliation countryCode="FR" type="organization" xml:id="embj201141-aff-0002"><orgName>Danisco France SAS, Dangé‐Saint‐Romain</orgName><address><country>France</country></address></affiliation><affiliation countryCode="US" type="organization" xml:id="embj201141-aff-0003"><orgName>Danisco USA Inc.</orgName><address><city>Madison</city><countryPart>WI</countryPart><country>USA</country></address></affiliation></affiliationGroup><keywordGroup type="author"><keyword xml:id="embj201141-kwd-0001">Cas3</keyword><keyword xml:id="embj201141-kwd-0002">CRISPR</keyword><keyword xml:id="embj201141-kwd-0003">Ecoli subtype</keyword><keyword xml:id="embj201141-kwd-0004">site‐directed mutagenesis</keyword><keyword xml:id="embj201141-kwd-0005"><i>Streptococcus thermophilus</i></keyword></keywordGroup><supportingInformation><supportingInfoItem xml:id="embj201141-sup-0001"><mediaResource alt="supplementary data" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201141:embj201141-sup-0001"></mediaResource><caption>Supplementary Figures S1–S5</caption></supportingInfoItem><supportingInfoItem xml:id="embj201141-sup-0002"><mediaResource alt="supplementary data" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201141:embj201141"></mediaResource><caption>Review Process File</caption></supportingInfoItem></supportingInformation><abstractGroup><abstract type="main"><p>Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In <i>Escherichia coli</i>, Cas3 is essential for crRNA‐guided interference with virus proliferation. Cas3 contains N‐terminal HD phosphohydrolase and C‐terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and <i>in vitro</i> functional analysis of the Cas3 protein of the <i>Streptococcus thermophilus</i> CRISPR4 (Ecoli subtype) system. Cas3 possesses a single‐stranded DNA (ssDNA)‐stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP‐independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation‐of‐function mutants (ATPase<sup>+</sup>/nuclease<sup>−</sup> and ATPase<sup>−</sup>/nuclease<sup>+</sup>) were obtained by site‐directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.</p></abstract><abstract type="short"><p>Cas3 is an essential protein of unknown function required for CRISPR‐based bacteriophage immunity in bacteria. Here, the biochemical activities of Cas3 are demonstrated and mechanistic implications for immunity are discussed.</p></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Cas3 nuclease/helicase</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system</title></titleInfo><name type="personal"><namePart type="given">Tomas</namePart><namePart type="family">Sinkunas</namePart><affiliation>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Giedrius</namePart><namePart type="family">Gasiunas</namePart><affiliation>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Christophe</namePart><namePart type="family">Fremaux</namePart><affiliation>Danisco France SAS, Dangé‐Saint‐Romain, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Rodolphe</namePart><namePart type="family">Barrangou</namePart><affiliation>Danisco USA Inc., WI, Madison, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Philippe</namePart><namePart type="family">Horvath</namePart><affiliation>Danisco France SAS, Dangé‐Saint‐Romain, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Virginijus</namePart><namePart type="family">Siksnys</namePart><affiliation>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania</affiliation><affiliation>E-mail: siksnys@ibt.lt</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</genre><originInfo><publisher>John Wiley & Sons, Ltd</publisher><place><placeTerm type="text">Chichester, UK</placeTerm></place><dateIssued encoding="w3cdtf">2011-04-06</dateIssued><dateCaptured encoding="w3cdtf">2010-11-06</dateCaptured><dateValid encoding="w3cdtf">2011-02-01</dateValid><copyrightDate encoding="w3cdtf">2011</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><abstract>Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA‐guided interference with virus proliferation. Cas3 contains N‐terminal HD phosphohydrolase and C‐terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single‐stranded DNA (ssDNA)‐stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP‐independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation‐of‐function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site‐directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.</abstract><abstract type="short">Cas3 is an essential protein of unknown function required for CRISPR‐based bacteriophage immunity in bacteria. Here, the biochemical activities of Cas3 are demonstrated and mechanistic implications for immunity are discussed.</abstract><note type="additional physical form">Supplementary Figures S1–S5Review Process File</note><subject><genre>keywords</genre><topic>Cas3</topic><topic>CRISPR</topic><topic>Ecoli subtype</topic><topic>site‐directed mutagenesis</topic><topic>Streptococcus thermophilus</topic></subject><relatedItem type="host"><titleInfo><title>The EMBO Journal</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><subject><genre>index-terms</genre><topic authorityURI="http://psi.embo.org/14602075/EMBO36">RNA Biology</topic><topic authorityURI="http://psi.embo.org/14602075/EMBO23">Microbiology, Virology & Host Pathogen Interaction</topic></subject><subject><genre>article-category</genre><topic>Article</topic><topic>Article</topic></subject><identifier type="ISSN">0261-4189</identifier><identifier type="eISSN">1460-2075</identifier><identifier type="DOI">10.1002/(ISSN)1460-2075</identifier><identifier type="PublisherID">EMBJ</identifier><part><date>2011</date><detail type="volume"><caption>vol.</caption><number>30</number></detail><detail type="issue"><caption>no.</caption><number>7</number></detail><extent unit="pages"><start>1335</start><end>1342</end><total>8</total></extent></part></relatedItem><relatedItem type="references" displayLabel="embj201141-citation-0001"><titleInfo><title>The HD domain defines a new superfamily of metal‐dependent phosphohydrolases</title></titleInfo><name type="personal"><namePart type="given">L</namePart><namePart type="family">Aravind</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">EV</namePart><namePart type="family">Koonin</namePart><role><roleTerm type="text">author</roleTerm></role></name><genre>journal-article</genre><note type="citation/reference">Aravind L, Koonin EV (1998) The HD domain defines a new superfamily of metal‐dependent phosphohydrolases. 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