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dc.contributor.advisorWhite, Malcolm F.
dc.contributor.authorAthukoralage, Januka Sahan
dc.description.abstractCRISPR-Cas systems provide prokaryotes with adaptative immunity from invading Mobile Genetic Elements (MGEs). Type III CRISPR-Cas systems consist of a multiprotein effector complex and CRISPR ancillary proteins; both essential for MGE elimination. In 2017, two groups independently identified that type III CRISPR-Cas complexes synthesised cyclic oligoadenylate (cOA) second messengers in response to detection of foreign RNA. cOA was made using ATP (adenosine triphosphate) and consisted of 3-6, 3’-5’ linked AMP subunits (cAn, n=3-6). cOA was found to activate CRISPR ancillary nucleases, which eliminated MGEs by cleaving nucleic acids non- specifically. CRISPR ancillary proteins are diverse, consisting of a cOA sensor domain fused to a toxin effector domain. This led to speculation that, if not controlled, collateral damage from the immune response could lead to cell dormancy or death, an unfavourable outcome for unicellular organisms. The work described herein details the identification of a new class of enzyme, termed “ring nuclease”, which degrades cyclic tetra-adenylate (cA4) second messengers and regulates the type III CRISPR immune response. The publications presented characterise five distinct ring nuclease families. These include the CRISPR ring nuclease 1 (Crn1) limited to the archaea, cOA activated self-inactivating CRISPR ancillary ribonucleases and the highly unusual Csx3/Crn3 family, which collectively extend ring nuclease distribution. Also presented are the anti-CRISPR DUF1874 family variant ring nucleases, which viruses employ to subvert type III CRISPR immunity, and the homologous Crn2 family found in association with type III CRISPR-Cas systems in prokaryotic genomes. Biochemical and biophysical characterisation of these proteins reveal diverse mechanisms underlying regulation of type III CRISPR defence, and kinetic modelling demonstrate the key roles of ring nucleases in governing the outcome of infections. These works provide fundamental insights into the regulation of a sophisticated, widespread and potent prokaryotic immune system, and select ring nucleases hold great promise for increasing the efficacy of bacteriophage therapies targeting pathogenic bacteria.en_US
dc.publisherUniversity of St Andrews
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 International*
dc.subjectCyclic oligoadenylateen_US
dc.titleKilling the messenger : discovery of enzymes degrading cyclic oligoadenylate defence activators synthesised by type III CRISPR immune systemsen_US
dc.contributor.sponsorRoyal Society (Great Britain)en_US
dc.contributor.sponsorBiotechnology and Biological Sciences Research Council (BBSRC)en_US
dc.contributor.sponsorUnited States. National Aeronautics and Space Administration (NASA)en_US
dc.contributor.sponsorWellcome Trusten_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US
dc.publisher.institutionThe University of St Andrewsen_US

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    Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
    Except where otherwise noted within the work, this item's licence for re-use is described as Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International