title: In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target.
abstract: Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. In Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.
authors.forename: Sabin
authors.initials: S
authors.lastname: Mulepati
authors.forename: Scott
authors.initials: S
authors.lastname: Bailey
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D007107:Immune System / Q000502:physiology*; D010957:Plasmids; D012329:RNA, Bacterial / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate
keywords: CRISPR; Cas3; Cascade; DNA; DNA Helicase; DNA-binding Protein; Nuclease; RNA; RNA-binding protein