| Structural highlights
Function
CAS6_ECOLI CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).[1] [2] [3] [4] [5] CasE is required to process the pre-crRNA into single repeat-spacer units, with an 8-nt 5'-repeat DNA tag that may help other proteins recognize the crRNA. This subunit alone will cleave pre-crRNA, as will CasCDE or CasCE; cleavage does not require divalent metals or ATP. CasCDE alone is also able to form R-loops. Partially inhibits the cleavage of Holliday junctions by YgbT (Cas1). Yields a 5'-hydroxy group and a 2',3'-cyclic phosphate terminus.[6] [7] [8] [9] [10] A component of Cascade, which participates in CRISPR interference, the third stage of CRISPR immunity. Cascade binds both crRNA and in a sequence-specific manner negatively supercoiled dsDNA target. This leads to the formation of an R-loop in which the crRNA binds the target DNA, displacing the noncomplementary strand. Cas3 is recruited to Cascade, nicks target DNA and then unwinds and cleaves the target, leading to DNA degradation and invader neutralization.[11] [12] [13] [14] [15]
Publication Abstract from PubMed
In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 A x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.
Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.,van Erp PB, Jackson RN, Carter J, Golden SM, Bailey S, Wiedenheft B Nucleic Acids Res. 2015 Aug 3. pii: gkv793. PMID:26243775[16]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008 Aug 15;321(5891):960-4. doi: 10.1126/science.1159689. PMID:18703739 doi:http://dx.doi.org/10.1126/science.1159689
- ↑ Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol Microbiol. 2011 Jan;79(2):484-502. doi:, 10.1111/j.1365-2958.2010.07465.x. Epub 2010 Dec 7. PMID:21219465 doi:10.1111/j.1365-2958.2010.07465.x
- ↑ Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol Microbiol. 2011 Feb;79(3):584-99. doi: 10.1111/j.1365-2958.2010.07482.x. Epub, 2010 Dec 13. PMID:21255106 doi:10.1111/j.1365-2958.2010.07482.x
- ↑ Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011 May;18(5):529-36. doi: 10.1038/nsmb.2019. Epub 2011 Apr, 3. PMID:21460843 doi:http://dx.doi.org/10.1038/nsmb.2019
- ↑ Howard JA, Delmas S, Ivancic-Bace I, Bolt EL. Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein. Biochem J. 2011 Oct 1;439(1):85-95. doi: 10.1042/BJ20110901. PMID:21699496 doi:http://dx.doi.org/10.1042/BJ20110901
- ↑ Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008 Aug 15;321(5891):960-4. doi: 10.1126/science.1159689. PMID:18703739 doi:http://dx.doi.org/10.1126/science.1159689
- ↑ Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol Microbiol. 2011 Jan;79(2):484-502. doi:, 10.1111/j.1365-2958.2010.07465.x. Epub 2010 Dec 7. PMID:21219465 doi:10.1111/j.1365-2958.2010.07465.x
- ↑ Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol Microbiol. 2011 Feb;79(3):584-99. doi: 10.1111/j.1365-2958.2010.07482.x. Epub, 2010 Dec 13. PMID:21255106 doi:10.1111/j.1365-2958.2010.07482.x
- ↑ Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011 May;18(5):529-36. doi: 10.1038/nsmb.2019. Epub 2011 Apr, 3. PMID:21460843 doi:http://dx.doi.org/10.1038/nsmb.2019
- ↑ Howard JA, Delmas S, Ivancic-Bace I, Bolt EL. Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein. Biochem J. 2011 Oct 1;439(1):85-95. doi: 10.1042/BJ20110901. PMID:21699496 doi:http://dx.doi.org/10.1042/BJ20110901
- ↑ Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008 Aug 15;321(5891):960-4. doi: 10.1126/science.1159689. PMID:18703739 doi:http://dx.doi.org/10.1126/science.1159689
- ↑ Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol Microbiol. 2011 Jan;79(2):484-502. doi:, 10.1111/j.1365-2958.2010.07465.x. Epub 2010 Dec 7. PMID:21219465 doi:10.1111/j.1365-2958.2010.07465.x
- ↑ Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol Microbiol. 2011 Feb;79(3):584-99. doi: 10.1111/j.1365-2958.2010.07482.x. Epub, 2010 Dec 13. PMID:21255106 doi:10.1111/j.1365-2958.2010.07482.x
- ↑ Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011 May;18(5):529-36. doi: 10.1038/nsmb.2019. Epub 2011 Apr, 3. PMID:21460843 doi:http://dx.doi.org/10.1038/nsmb.2019
- ↑ Howard JA, Delmas S, Ivancic-Bace I, Bolt EL. Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein. Biochem J. 2011 Oct 1;439(1):85-95. doi: 10.1042/BJ20110901. PMID:21699496 doi:http://dx.doi.org/10.1042/BJ20110901
- ↑ van Erp PB, Jackson RN, Carter J, Golden SM, Bailey S, Wiedenheft B. Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. Nucleic Acids Res. 2015 Aug 3. pii: gkv793. PMID:26243775 doi:http://dx.doi.org/10.1093/nar/gkv793
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