PMID:21343909

Citation	Sinkunas, T, Gasiunas, G, Fremaux, C, Barrangou, R, Horvath, P and Siksnys, V (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30:1335-42
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.
Links	PubMed PMC3094125 Online version:10.1038/emboj.2011.41
Keywords	Adenosine Triphosphate/metabolism; Cloning, Molecular; DNA Helicases/genetics; DNA Helicases/metabolism; DNA Mutational Analysis; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; DNA, Single-Stranded/metabolism; Escherichia coli/genetics; Gene Expression; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Streptococcus thermophilus/enzymology; Streptococcus thermophilus/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STRTR:CAS3	enables	GO:0004536: deoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
STRTR:CAS3		GO:0004536: deoxyribonuclease activity	ECO:0000314:		F		Figure 3 (A) shows the nuclease activity of Cas3 in the presence of MG2+ ions. Incubating Cas3 with M13mp18 ssDNA over a two hour period shows increased levels of Cas3 correspond to increased ssDNA degradation.	complete CACAO 7119

References

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