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dc.contributor.advisorWhite, Malcolm F.
dc.contributor.authorAthukoralage, Januka Sahan
dc.coverage.spatial315en_US
dc.date.accessioned2024-03-05T11:29:36Z
dc.date.available2024-03-05T11:29:36Z
dc.date.issued2021-06-30
dc.identifier.urihttps://hdl.handle.net/10023/29428
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.language.isoenen_US
dc.publisherUniversity of St Andrews
dc.relationRouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S., & White, M. F. (2018). Control of cyclic oligoadenylate synthesis in a type III CRISPR system. eLife, 7, Article e36734. https://doi.org/10.7554/eLife.36734en
dc.relation
dc.relationAthukoralage, J. S., Rouillon, C., Graham, S., Grüschow, S., & White, M. F. (2018). Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature, 562(7726), 277-280. https://doi.org/10.1038/s41586-018-0557-5en
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dc.relationRouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S., & White, M. F. (2019). Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems. In S. Bailey (Ed.), Methods in Enzymology (pp. 191-218). (Methods in Enzymology; Vol. 616). Academic Press/Elsevier . https://doi.org/10.1016/bs.mie.2018.10.020en
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dc.relationAthukoralage, J. S., Graham, S., Grüschow, S., Rouillon, C., & White, M. F. (2019). A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator. Journal of Molecular Biology, 431(15), 2894-2899. https://doi.org/10.1016/j.jmb.2019.04.041en
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dc.relationAthukoralage, J. S., McMahon, S., Zhang, C., Gruschow, S., Graham, S., Krupovic, M., Whitaker, R., Gloster, T., & White, M. (2020). An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature, 577(7791), 572-575. https://doi.org/10.1038/s41586-019-1909-5en
dc.relation
dc.relationAthukoralage, J. S., Graham, S., Rouillon, C., Gruschow, S., M Czekster, C., & White, M. (2020). The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling. eLife, 9, Article e55852. https://doi.org/10.7554/eLife.55852en
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dc.relationSamolygo, A., Athukoralage, J. S., Graham, S., & White, M. (2020). Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence. Nucleic Acids Research, 48(11), 6149–6156. https://doi.org/10.1093/nar/gkaa298en
dc.relation
dc.relationAthukoralage, J. S., McQuarrie, S. J., Gruschow, S., Graham, S., Gloster, T., & White, M. (2020). Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage. eLife, 9, Article e57627. https://doi.org/10.7554/eLife.57627en
dc.relation.urihttps://doi.org/10.7554/eLife.36734
dc.relation.urihttps://doi.org/10.1038/s41586-018-0557-5
dc.relation.urihttps://doi.org/10.1016/bs.mie.2018.10.020
dc.relation.urihttps://doi.org/10.1016/j.jmb.2019.04.041
dc.relation.urihttps://doi.org/10.1038/s41586-019-1909-5
dc.relation.urihttps://doi.org/10.7554/eLife.55852
dc.relation.urihttps://doi.org/10.1093/nar/gkaa298
dc.relation.urihttps://doi.org/10.7554/eLife.57627
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/*
dc.subjectCRISPR-Casen_US
dc.subjectCyclic oligoadenylateen_US
dc.subjectBiologyen_US
dc.subjectPhageen_US
dc.subjectBacteriaen_US
dc.subjectArchaeaen_US
dc.subjectVirusesen_US
dc.subjectEnzymologyen_US
dc.titleKilling the messenger : discovery of enzymes degrading cyclic oligoadenylate defence activators synthesised by type III CRISPR immune systemsen_US
dc.typeThesisen_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.qualificationlevelDoctoralen_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US
dc.publisher.institutionThe University of St Andrewsen_US
dc.identifier.doihttps://doi.org/10.17630/sta/806
dc.identifier.grantnumberBB/M000400/1en_US
dc.identifier.grantnumberBB/M021017/1en_US
dc.identifier.grantnumberBB/S000313/1en_US
dc.identifier.grantnumberBB/R008035/1en_US
dc.identifier.grantnumberBB/T004789/1en_US
dc.identifier.grantnumberNNX14AK23Gen_US


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