title: Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.
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.
authors.affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Sinkunas
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.forename: Christophe
authors.initials: C
authors.lastname: Fremaux
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.forename: Philippe
authors.initials: P
authors.lastname: Horvath
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D048249:Streptococcus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004265:DNA Helicases