Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system
Identifieur interne : 002583 ( Main/Exploration ); précédent : 002582; suivant : 002584Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system
Auteurs : Tomas Sinkunas [Lituanie] ; Giedrius Gasiunas [Lituanie] ; Christophe Fremaux [France] ; Rodolphe Barrangou [États-Unis] ; Philippe Horvath [France] ; Virginijus Siksnys [Lituanie]Source :
- 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
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Istex, to step Corpus: 001C53
- to stream Istex, to step Curation: 001C53
- to stream Istex, to step Checkpoint: 000512
- to stream Main, to step Merge: 002608
- to stream Main, to step Curation: 002583
Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system</title>
<author><name sortKey="Sinkunas, Tomas" sort="Sinkunas, Tomas" uniqKey="Sinkunas T" first="Tomas" last="Sinkunas">Tomas Sinkunas</name>
</author>
<author><name sortKey="Gasiunas, Giedrius" sort="Gasiunas, Giedrius" uniqKey="Gasiunas G" first="Giedrius" last="Gasiunas">Giedrius Gasiunas</name>
</author>
<author><name sortKey="Fremaux, Christophe" sort="Fremaux, Christophe" uniqKey="Fremaux C" first="Christophe" last="Fremaux">Christophe Fremaux</name>
</author>
<author><name sortKey="Barrangou, Rodolphe" sort="Barrangou, Rodolphe" uniqKey="Barrangou R" first="Rodolphe" last="Barrangou">Rodolphe Barrangou</name>
</author>
<author><name sortKey="Horvath, Philippe" sort="Horvath, Philippe" uniqKey="Horvath P" first="Philippe" last="Horvath">Philippe Horvath</name>
</author>
<author><name sortKey="Siksnys, Virginijus" sort="Siksnys, Virginijus" uniqKey="Siksnys V" first="Virginijus" last="Siksnys">Virginijus Siksnys</name>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">ISTEX</idno>
<idno type="RBID">ISTEX:7276EDF70BEF82B30A53825E4F8FF243B80B98EB</idno>
<date when="2011" year="2011">2011</date>
<idno type="doi">10.1038/emboj.2011.41</idno>
<idno type="url">https://api.istex.fr/ark:/67375/WNG-TCWFBFS5-V/fulltext.pdf</idno>
<idno type="wicri:Area/Istex/Corpus">001C53</idno>
<idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001C53</idno>
<idno type="wicri:Area/Istex/Curation">001C53</idno>
<idno type="wicri:Area/Istex/Checkpoint">000512</idno>
<idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">000512</idno>
<idno type="wicri:doubleKey">0261-4189:2011:Sinkunas T:cas:is:a</idno>
<idno type="wicri:Area/Main/Merge">002608</idno>
<idno type="wicri:Area/Main/Curation">002583</idno>
<idno type="wicri:Area/Main/Exploration">002583</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title level="a" type="main">Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system</title>
<author><name sortKey="Sinkunas, Tomas" sort="Sinkunas, Tomas" uniqKey="Sinkunas T" first="Tomas" last="Sinkunas">Tomas Sinkunas</name>
<affiliation wicri:level="1"><country xml:lang="fr">Lituanie</country>
<wicri:regionArea>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius</wicri:regionArea>
<wicri:noRegion>Vilnius</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Gasiunas, Giedrius" sort="Gasiunas, Giedrius" uniqKey="Gasiunas G" first="Giedrius" last="Gasiunas">Giedrius Gasiunas</name>
<affiliation wicri:level="1"><country xml:lang="fr">Lituanie</country>
<wicri:regionArea>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius</wicri:regionArea>
<wicri:noRegion>Vilnius</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Fremaux, Christophe" sort="Fremaux, Christophe" uniqKey="Fremaux C" first="Christophe" last="Fremaux">Christophe Fremaux</name>
<affiliation wicri:level="1"><country xml:lang="fr">France</country>
<wicri:regionArea>Danisco France SAS, Dangé‐Saint‐Romain</wicri:regionArea>
<wicri:noRegion>Dangé‐Saint‐Romain</wicri:noRegion>
<wicri:noRegion>Dangé‐Saint‐Romain</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Barrangou, Rodolphe" sort="Barrangou, Rodolphe" uniqKey="Barrangou R" first="Rodolphe" last="Barrangou">Rodolphe Barrangou</name>
<affiliation wicri:level="1"><country xml:lang="fr">États-Unis</country>
<wicri:regionArea>Danisco USA Inc., WI, Madison</wicri:regionArea>
<wicri:noRegion>Madison</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Horvath, Philippe" sort="Horvath, Philippe" uniqKey="Horvath P" first="Philippe" last="Horvath">Philippe Horvath</name>
<affiliation wicri:level="1"><country xml:lang="fr">France</country>
<wicri:regionArea>Danisco France SAS, Dangé‐Saint‐Romain</wicri:regionArea>
<wicri:noRegion>Dangé‐Saint‐Romain</wicri:noRegion>
<wicri:noRegion>Dangé‐Saint‐Romain</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Siksnys, Virginijus" sort="Siksnys, Virginijus" uniqKey="Siksnys V" first="Virginijus" last="Siksnys">Virginijus Siksnys</name>
<affiliation wicri:level="1"><country xml:lang="fr">Lituanie</country>
<wicri:regionArea>Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius</wicri:regionArea>
<wicri:noRegion>Vilnius</wicri:noRegion>
</affiliation>
<affiliation wicri:level="1"><country wicri:rule="url">Lituanie</country>
</affiliation>
</author>
</analytic>
<monogr></monogr>
<series><title level="j" type="main">The EMBO Journal</title>
<title level="j" type="alt">THE EMBO JOURNAL</title>
<idno type="ISSN">0261-4189</idno>
<idno type="eISSN">1460-2075</idno>
<imprint><biblScope unit="vol">30</biblScope>
<biblScope unit="issue">7</biblScope>
<biblScope unit="page" from="1335">1335</biblScope>
<biblScope unit="page" to="1342">1342</biblScope>
<biblScope unit="page-count">8</biblScope>
<publisher>John Wiley & Sons, Ltd</publisher>
<pubPlace>Chichester, UK</pubPlace>
<date type="published" when="2011-04-06">2011-04-06</date>
</imprint>
<idno type="ISSN">0261-4189</idno>
</series>
</biblStruct>
</sourceDesc>
<seriesStmt><idno type="ISSN">0261-4189</idno>
</seriesStmt>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="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.</div>
<div type="abstract">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.</div>
</front>
</TEI>
<affiliations><list><country><li>France</li>
<li>Lituanie</li>
<li>États-Unis</li>
</country>
</list>
<tree><country name="Lituanie"><noRegion><name sortKey="Sinkunas, Tomas" sort="Sinkunas, Tomas" uniqKey="Sinkunas T" first="Tomas" last="Sinkunas">Tomas Sinkunas</name>
</noRegion>
<name sortKey="Gasiunas, Giedrius" sort="Gasiunas, Giedrius" uniqKey="Gasiunas G" first="Giedrius" last="Gasiunas">Giedrius Gasiunas</name>
<name sortKey="Siksnys, Virginijus" sort="Siksnys, Virginijus" uniqKey="Siksnys V" first="Virginijus" last="Siksnys">Virginijus Siksnys</name>
<name sortKey="Siksnys, Virginijus" sort="Siksnys, Virginijus" uniqKey="Siksnys V" first="Virginijus" last="Siksnys">Virginijus Siksnys</name>
</country>
<country name="France"><noRegion><name sortKey="Fremaux, Christophe" sort="Fremaux, Christophe" uniqKey="Fremaux C" first="Christophe" last="Fremaux">Christophe Fremaux</name>
</noRegion>
<name sortKey="Horvath, Philippe" sort="Horvath, Philippe" uniqKey="Horvath P" first="Philippe" last="Horvath">Philippe Horvath</name>
</country>
<country name="États-Unis"><noRegion><name sortKey="Barrangou, Rodolphe" sort="Barrangou, Rodolphe" uniqKey="Barrangou R" first="Rodolphe" last="Barrangou">Rodolphe Barrangou</name>
</noRegion>
</country>
</tree>
</affiliations>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002583 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 002583 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= MersV1 |flux= Main |étape= Exploration |type= RBID |clé= ISTEX:7276EDF70BEF82B30A53825E4F8FF243B80B98EB |texte= Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system }}
![]() | This area was generated with Dilib version V0.6.33. | ![]() |