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Mechanism of CRISPR-Cas Immunologica...

Nunez, James Karlo.

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Mechanism of CRISPR-Cas Immunological Memory.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Mechanism of CRISPR-Cas Immunological Memory./
作者:	Nunez, James Karlo.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2016,
面頁冊數:	120 p.
附註:	Source: Dissertation Abstracts International, Volume: 77-12(E), Section: B.
Contained By:	Dissertation Abstracts International77-12B(E).
標題:	Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10150950
ISBN:	9781369057140

Mechanism of CRISPR-Cas Immunological Memory.
Nunez, James Karlo.

Mechanism of CRISPR-Cas Immunological Memory. - Ann Arbor : ProQuest Dissertations & Theses, 2016 - 120 p.

Source: Dissertation Abstracts International, Volume: 77-12(E), Section: B.

Thesis (Ph.D.)--University of California, Berkeley, 2016.

Immunological memory, defined as the ability for cells to rapidly respond to previously encountered pathogens, has long been thought to be present solely in eukaryotes. Recently, it was discovered that bacteria and archaea also possess analogous adaptive immune systems called CRISPR--Cas ( clustered regularly interspaced short palindromic repeats-- CRISPR associated). CRISPRs are distinct genomic loci comprising of repeating 20-50 base pair (bp) sequences that are separated by foreign DNA-derived segments called spacers. Upon viral infection, a ~30 bp fragment of the foreign DNA is acquired into the CRISPR locus as a new spacer, serving as a molecular memory of that specific infection. The CRISPR RNA (crRNA) products of CRISPR loci are used as guides by Cas proteins to silence infectious foreign genetic elements containing sequence complementarity to the crRNA. The research presented in this dissertation elucidates the mechanisms underlying spacer acquisition during CRISPR immunity.

ISBN: 9781369057140Subjects--Topical Terms:

518028
Biochemistry.

Mechanism of CRISPR-Cas Immunological Memory.
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520 $a Immunological memory, defined as the ability for cells to rapidly respond to previously encountered pathogens, has long been thought to be present solely in eukaryotes. Recently, it was discovered that bacteria and archaea also possess analogous adaptive immune systems called CRISPR--Cas ( clustered regularly interspaced short palindromic repeats-- CRISPR associated). CRISPRs are distinct genomic loci comprising of repeating 20-50 base pair (bp) sequences that are separated by foreign DNA-derived segments called spacers. Upon viral infection, a ~30 bp fragment of the foreign DNA is acquired into the CRISPR locus as a new spacer, serving as a molecular memory of that specific infection. The CRISPR RNA (crRNA) products of CRISPR loci are used as guides by Cas proteins to silence infectious foreign genetic elements containing sequence complementarity to the crRNA. The research presented in this dissertation elucidates the mechanisms underlying spacer acquisition during CRISPR immunity.
520 $a Cas1 and Cas2, the only universally conserved Cas proteins present in CRISPR-harboring organisms, are sufficient to elicit spacer acquisition in E. coli. Using a combination of biochemical, bacterial genetics and X-ray crystallography experiments, we reveal the requirement of Cas1 and Cas2 to associate as a protein complex during CRISPR immunity. Cas1 is the catalytic subunit, whereas Cas2 functions as a scaffold within the complex. By determining crystal structures of Cas1--Cas2 bound to a 33 bp DNA from M13 bacteriophage, we uncover Cas1--Cas2 as a molecular ruler that precisely measures the foreign DNA across the entire complex with each DNA end terminating in the catalytic Cas1 active sites. Collectively, our structural data uncover how Cas1--Cas2 captures, measures and prepares foreign DNA for insertion into the CRISPR locus.
520 $a A hallmark of CRISPR immunological memory is the addition of foreign DNA spacers into CRISPR loci. By developing an in vitro assay to study spacer acquisition, we reveal that purified Cas1--Cas2 can be loaded with 18-50 bp spacer DNA for integration into a plasmid encoding the CRISPR locus. Cas1--Cas2 integrates spacers adjacent to the CRISPR repeats via a nucleophilic reaction of the DNA 3'--OH ends into the target DNA, in a mechanism similar to many retroviral integrases and transposases. Furthermore, the integration host factor (IHF) protein in E. coli binds the CRISPR leader to induce sufficient DNA bending required for the integration reaction, explaining the mechanistic basis for leader-proximal spacer acquisition observed in vivo. Together, these in vitro assays fully recapitulate immunological memory during prokaryotic CRISPR immunity.
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