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My work on Wikipedia

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My main focus was to add information about how CRISPR integrates new spacers in E. Coli. The CRISPR page had good information about the Ca1-Cas2 complex and its responsibility in integration new spacers. They did not have any mention that in E. Coli IHF is responsible for the chronological addition of new spacers so I added that. I then searched to see if there was any page on IHF. What I found was a page for bacterial DNA binding proteins. On this page there was a mention of what IHF is, but had no information on its role in some CRISPR systems. I added a brief addition describing how it is used in CRISPR, and slightly re organized the section so it made more sense. Unfortunately I wasn't able to add too much more as there is still a lot unknown about how this process is carried out, this is partially why my proposal is very relevant!

Spacer acquisition

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When a microbe is invaded by a virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in all three types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response.[1][2][3][4][5]

Multiple Cas1 proteins have been characterised and their structures resolved.[6][7][8] Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner.[9] Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA-[10] or (double strand) dsDNA-[11][12] specific endoribonuclease activity.

In the I-E system of E. coli Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers.[13] In this complex Cas2 performs a non-enzymatic scaffolding role,[13] binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays.[14][15][16] A key part of this system is that new spacers are always added at the begining of the CRISPR next to the leader sequence. In E. Coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration[17].

Bac_DNA_binding
anabaena hu-dna cocrystal structure (ahu6)
Identifiers
SymbolBac_DNA_binding
PfamPF00216
InterProIPR000119
PROSITEPDOC00044
SCOP21hue / SCOPe / SUPFAM
CDDcd00591
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

IHF

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Integration host factor, IHF, is a nucleoid-associated protein only found in gram negative bacteria[18]. It is a 20 kDa heterodimer, composed of α and β subunits that bind to the sequence 5' - WATCAANNNNTTR - 3' and bends the DNA approximately 160 degrees[19]. The β arms of IHF have Proline residues that help stabilize the DNA kinks. These kinks can help compact DNA and allow for supercoiling. The mode of binding to DNA depends on environmental factors, such as the concentration of ions present. With a high concentration of KCl, there is weak DNA bending. It has been found that sharper DNA bending occurs when the concentration of KCl is less than 100 mM, and IHF is not concentrated.[20]

IHF was discovered as a necessary co-factor for recombination of λ phage in to E.coli. In 2016 it was discovered that IHF also plays a key role in CRISPR type I and type II systems. It has a major role in allowing the Cas1-Cas2 complex to integrate new spacers into the CRISPR sequence. The bending of the DNA by IHF is thought to alter spacing in the DNA major and minor grooves, allowing the Cas1-Cas2 complex to make contact with the DNA bases[21]. This is a key function in the CRISPR system as it ensures that new spacers area always added at the beginning of the CRISPE sequence next to the leader sequence. This directing of integration by IHF ensures that spacers are added chronologically, allowing better protection against the most recent viral infection[22].

References

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  1. ^ Aliyari R, Ding SW (January 2009). "RNA-based viral immunity initiated by the Dicer family of host immune receptors". Immunological Reviews. 227 (1): 176–88. doi:10.1111/j.1600-065X.2008.00722.x. PMC 2676720. PMID 19120484.
  2. ^ Dugar G, Herbig A, Förstner KU, Heidrich N, Reinhardt R, Nieselt K, Sharma CM (May 2013). "High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates". PLoS Genetics. 9 (5): e1003495. doi:10.1371/journal.pgen.1003495. PMC 3656092. PMID 23696746.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  3. ^ Hatoum-Aslan A, Maniv I, Marraffini LA (December 2011). "Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site". Proceedings of the National Academy of Sciences of the United States of America. 108 (52): 21218–22. Bibcode:2011PNAS..10821218H. doi:10.1073/pnas.1112832108. PMC 3248500. PMID 22160698.
  4. ^ Yosef I, Goren MG, Qimron U (July 2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli". Nucleic Acids Research. 40 (12): 5569–76. doi:10.1093/nar/gks216. PMC 3384332. PMID 22402487.
  5. ^ Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012). "CRISPR interference directs strand specific spacer acquisition". PloS One. 7 (4): e35888. Bibcode:2012PLoSO...735888S. doi:10.1371/journal.pone.0035888. PMC 3338789. PMID 22558257.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  6. ^ 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 (January 2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair". Molecular Microbiology. 79 (2): 484–502. doi:10.1111/j.1365-2958.2010.07465.x. PMC 3071548. PMID 21219465.
  7. ^ Han D, Lehmann K, Krauss G (June 2009). "SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA". FEBS Letters. 583 (12): 1928–32. doi:10.1016/j.febslet.2009.04.047. PMID 19427858.
  8. ^ Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA (June 2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense". Structure. 17 (6): 904–12. doi:10.1016/j.str.2009.03.019. PMID 19523907.
  9. ^ Cite error: The named reference pmid22337052 was invoked but never defined (see the help page).
  10. ^ Beloglazova N, Brown G, Zimmerman MD, Proudfoot M, Makarova KS, Kudritska M, Kochinyan S, Wang S, Chruszcz M, Minor W, Koonin EV, Edwards AM, Savchenko A, Yakunin AF (July 2008). "A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats". The Journal of Biological Chemistry. 283 (29): 20361–71. doi:10.1074/jbc.M803225200. PMC 2459268. PMID 18482976.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  11. ^ Samai P, Smith P, Shuman S (December 2010). "Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris". Acta Crystallographica Section F. 66 (Pt 12): 1552–6. doi:10.1107/S1744309110039801. PMC 2998353. PMID 21139194.
  12. ^ Nam KH, Ding F, Haitjema C, Huang Q, DeLisa MP, Ke A (October 2012). "Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein". The Journal of Biological Chemistry. 287 (43): 35943–52. doi:10.1074/jbc.M112.382598. PMC 3476262. PMID 22942283.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  13. ^ a b Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA (June 2014). "Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity". Nature Structural & Molecular Biology. 21 (6): 528–34. doi:10.1038/nsmb.2820. PMC 4075942. PMID 24793649.
  14. ^ Nuñez JK, Lee AS, Engelman A, Doudna JA (March 2015). "Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity". Nature. 519 (7542): 193–8. doi:10.1038/nature14237. PMC 4359072. PMID 25707795.
  15. ^ Wang J, Li J, Zhao H, Sheng G, Wang M, Yin M, Wang Y (November 2015). "Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems". Cell. 163 (4): 840–53. doi:10.1016/j.cell.2015.10.008. PMID 26478180.
  16. ^ Nuñez JK, Harrington LB, Kranzusch PJ, Engelman AN, Doudna JA (November 2015). "Foreign DNA capture during CRISPR-Cas adaptive immunity". Nature. 527 (7579): 535–8. doi:10.1038/nature15760. PMID 26503043.
  17. ^ Nuñez, James K.; Bai, Lawrence; Harrington, Lucas B.; Hinder, Tracey L.; Doudna, Jennifer A. (2016-06-16). "CRISPR Immunological Memory Requires a Host Factor for Specificity". Molecular Cell. 62 (6): 824–833. doi:10.1016/j.molcel.2016.04.027. ISSN 1097-4164. PMID 27211867.
  18. ^ Dillon, Shane C.; Dorman, Charles J. (2010-03-01). "Bacterial nucleoid-associated proteins, nucleoid structure and gene expression". Nature Reviews Microbiology. 8 (3): 185–195. doi:10.1038/nrmicro2261. ISSN 1740-1526.
  19. ^ Nuñez, James K.; Bai, Lawrence; Harrington, Lucas B.; Hinder, Tracey L.; Doudna, Jennifer A. (2016-06-16). "CRISPR Immunological Memory Requires a Host Factor for Specificity". Molecular Cell. 62 (6): 824–833. doi:10.1016/j.molcel.2016.04.027. ISSN 1097-4164. PMID 27211867.
  20. ^ Lin, J.; Chen, H.; Droge, P.; Yan, J. (2012). "Physical Organization of DNA by Multiple Non-Specific DNA-Binding Modes of Integration Host Factor (IHF)". PLoS ONE. 7 (11): e49885. doi:10.1371/journal.pone.0049885. PMC 3498176. PMID 23166787.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  21. ^ Nuñez, James K.; Bai, Lawrence; Harrington, Lucas B.; Hinder, Tracey L.; Doudna, Jennifer A. (2016-06-16). "CRISPR Immunological Memory Requires a Host Factor for Specificity". Molecular Cell. 62 (6): 824–833. doi:10.1016/j.molcel.2016.04.027. ISSN 1097-4164. PMID 27211867.
  22. ^ Sorek, Rotem; Lawrence, C. Martin; Wiedenheft, Blake. "CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea". Annual Review of Biochemistry. 82 (1): 237–266. doi:10.1146/annurev-biochem-072911-172315.
This article incorporates text from the public domain Pfam and InterPro: IPR000119


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