title: A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Natalia
authors.initials: N
authors.lastname: Beloglazova
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Zimmerman
authors.forename: Michael
authors.initials: M
authors.lastname: Proudfoot
authors.forename: Kira S
authors.initials: KS
authors.lastname: Makarova
authors.forename: Marina
authors.initials: M
authors.lastname: Kudritska
authors.forename: Samvel
authors.initials: S
authors.lastname: Kochinyan
authors.forename: Shuren
authors.initials: S
authors.lastname: Wang
authors.forename: Maksymilian
authors.initials: M
authors.lastname: Chruszcz
authors.forename: Wladek
authors.initials: W
authors.lastname: Minor
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
authors.forename: Aled M
authors.initials: AM
authors.lastname: Edwards
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D012313:RNA; D004722:Endoribonucleases