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:
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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. 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)
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