title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.
abstract: CRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
authors: Joshua R
authors: JR
authors: Elmore
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
authors: Nolan F
authors: NF
authors: Sheppard
authors: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32313, USA;
authors: Nancy
authors: N
authors: Ramia
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
authors: Trace
authors: T
authors: Deighan
authors: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32313, USA;
authors: Hong
authors: H
authors: Li
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA;
authors: Rebecca M
authors: RM
authors: Terns
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA; Department of Genetics, Florida State University, Tallahassee, Florida 32313, USA; Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
authors: Michael P
authors: MP
authors: Terns
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D020868:Gene Silencing / Q000502:physiology*; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D020027:Pyrococcus furiosus / Q000502:physiology; D012313:RNA / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012313:RNA; D003851:Deoxyribonucleases
keywords: CRISPR–Cas; Cmr; DNA nuclease; PAM; RNAi