Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.

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  • Additional Information
    • Source:
      Publisher: Academic Press Country of Publication: United States NLM ID: 0212271 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1557-7988 (Electronic) Linking ISSN: 00766879 NLM ISO Abbreviation: Methods Enzymol Subsets: MEDLINE
    • Publication Information:
      Original Publication: New York, Academic Press.
    • Subject Terms:
      CRISPR-Cas Systems*; Adenine Nucleotides/*metabolism ; Archaeal Proteins/*metabolism ; CRISPR-Associated Proteins/*metabolism ; Oligoribonucleotides/*metabolism ; Sulfolobus solfataricus/*metabolism; Adenine Nucleotides/genetics ; Archaeal Proteins/genetics ; CRISPR-Associated Proteins/genetics ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Kinetics ; Oligoribonucleotides/genetics ; Second Messenger Systems ; Signal Transduction ; Sulfolobus solfataricus/genetics
    • Abstract:
      Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.
      (© 2019 Elsevier Inc. All rights reserved.)
    • Grant Information:
      BB/M000400/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
    • Contributed Indexing:
      Keywords: CARF; CRISPR type III; Csm6; Csx1; Cyclic oligoadenylate; HEPN; MazEF; Ribonuclease; Ring nuclease
    • Accession Number:
      0 (Adenine Nucleotides)
      0 (Archaeal Proteins)
      0 (CRISPR-Associated Proteins)
      0 (Oligoribonucleotides)
      61172-40-5 (2',5'-oligoadenylate)
    • Publication Date:
      Date Created: 20190130 Date Completed: 20191114 Latest Revision: 20240306
    • Publication Date:
      20240306
    • Accession Number:
      10.1016/bs.mie.2018.10.020
    • Accession Number:
      30691643