Dokument: Untersuchungen zur Funktion des CRISPR/Cas-Systems von E. coli: Isolierung und Charakterisierung spezifischer Komponenten des Cascade-Komplexes und Aufbau eines Systems zur Spacer Integration
| Titel: | Untersuchungen zur Funktion des CRISPR/Cas-Systems von E. coli: Isolierung und Charakterisierung spezifischer Komponenten des Cascade-Komplexes und Aufbau eines Systems zur Spacer Integration | |||||||
| Weiterer Titel: | Studies on the function of the CRISPR/Cas system of E. coli: isolation and characterization of specific components of the cascade complex and establishment of a system for spacer integration | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=29981 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20140711-090310-2 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dipl.-Biol. Hermanns, Veronica [Autor] | |||||||
| Dateien: |
| |||||||
| Beitragende: | Prof. Dr. Wagner, Rolf [Betreuer/Doktorvater] Prof. Dr. Martin, William [Gutachter] | |||||||
| Stichwörter: | CRISPR, E. coli, spacer integration | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | Eine besonders wirkungsvolle Art der Immunabwehr in Prokaryoten ist das als CRISPR/Cas bezeichnete System, was für Clustered Regularly Interspaced Short Palindromic Repeats und den CRISPR associated proteins steht. Es verleiht Bakterien eine adaptive und vererbbare Immunität gegenüber Infektionen durch Fremd-DNA oder –RNA in Form von Viren oder Phagen. Aufgebaut ist es aus Subtyp-spezifischen Cas-Proteinen und einem nicht-kodierenden DNA-Bereich, dem CRISPR-Array. Dieser umfasst Regionen im Wirts-Genom, die aus palindromischen Sequenzwiederholungen (Repeats) bestehen und von variablen Spacer-Bereichen unterbrochen werden. Nach einer Erstinfektion der Bakterienzelle wird die fremde DNA als solche erkannt und von den Cas-Proteinen in kleine DNA-Fragmente gespalten. Mit dem Einbau dieser Fremd-DNA als neuer Spacer in den eigenen CRISPR-Array erfolgt die Immunisierung der Zelle. Für diesen, auch als Adaptation bezeichneten Mechanismus, sind neben den zwei ubiquitären Cas-Nukleasen Cas1 und Cas2 die Präsenz einer Leaderregion, sowie einer Repeat-Einheit essentielle Bestandteile.
In dieser Arbeit wurden Untersuchungen zur Aufklärung des Mechanismus der Spacerintegration in E. coli durchgeführt. Der verwendete Bakterienstamm BL21 AI besitzt einen CRISPR II Array, dem die für die Immunisierung essentiellen cas-Gene fehlen. Für eine detaillierte Charakterisierung des Adaptationsmechanismus stand die Entwicklung eines Plasmid-basierten Integrations-Assays im Vordergrund. Dazu musste zunächst ein Plasmid konstruiert werden, dass neben den fehlenden cas Genen, einen synthetischen CRISPR-Array trägt und als Grundlage für den Aufbau eines Plasmid-Systems zur Spacerintegration dient. Die erfolgreiche Etablierung des Plasmid-Systems konnte durch PCR-Amplifikationsstudien, die in vivo den Einbau von neuen Spacern in den plasmidären CRISPR-Array zeigten, verifiziert werden. Zudem konnten neu integrierte Spacersequenzen mittels single-colony-PCR (sc-PCR) identifiziert und analysiert werden. Die Einführung gezielter Punktmutationen innerhalb der ersten Repeatsequenz zeigte zum ersten Mal eine sequenzspezifische Spaltung durch die Cas1 und Cas2 Proteine. In weiterführenden Southern Blot Analysen konnte die Hypothese eines sequenzspezifisch-, versetzten Schnittes an beiden Strängen der ersten Repeatsequenz in vivo bestätigt werden und die Bildung von Reaktions-Intermediaten (Zwischenstufen) detektiert werden. Die Analyse dieser Intermediate weist auf eine gekoppelte Spaltungs-Ligations-Reaktion während der Integration hin. In einem weiteren Teil dieser Arbeit wurden drei der fünf den Cascade-Komplex bildenden Cas-Proteine (Cse1, Cse2 und Cas7) affinitätschromatographisch aufgereinigt und durch in vitro Bindestudien mit doppel- und einzelsträngigen Nukleinsäuren analysiert. Dabei konnte eine Bindung des Cse1-Proteins nicht nur an kurze doppelsträngige und einzelsträngige DNA, sondern auch mit einer hohen Affinität an pre-crRNA von E. coli gezeigt werden. Das Cse2 Protein hingegen zeigte eine selektive Bindung an Einzelstrang-DNA.A particularly effective type of immune system in prokaryotes is known as CRISPR/Cas system, which stands for Clustered Regularly Spaced Interspaced Short Palindromic Repeats and the CRISPR associated proteins. It provides prokaryotes with an adaptive and heritable immunity against infection by foreign DNA or RNA (e.g. phages or plasmids). The system consists of subtype-specific Cas proteins and a non coding DNA region, termed CRISPR array consisting of palindromic repeats that are interspaced by variable spacer sequences. After initial infection of the bacterial cell the invading DNA is recognized and cleaved by Cas proteins into small DNA fragments. Integration of these foreign DNA-derived fragments into the CRISPR array as new spacers results in the immunization of the cell. This process, also referred as adaptation, depends on the presence of the two ubiquitous nucleases Cas1 and Cas2 but also on the presence of a leader region followed by at least one repeat sequence. To elucidate the mechanism of spacer integration in E. coli in more detail, in vivo spacer acquisition assays were performed using E. coli BL21 AI. This bacterial strain contains a CRISPR II array but lacks all cas genes, including cas1 and cas2, which are required for the immunization. In order to study the mechanism of the adaptation stage, it was necessary to develop a plasmid-based integration assay. Therefore, a plasmid was constructed, which contains the essential cas1 and cas2 genes and a synthetic CRISPR array. The acquisition of new spacers into the plasmid-located array could be verified by in vivo assays. Sequences of the newly aquired spacers were determined by single-colony PCR reactions (sc-PCR). Selective point mutations within the first repeat sequence were introduced and analyzed for their effects on spacer aquisition. The obtained results demonstrated a sequence-specific cleavage by the Cas1 and Cas2 proteins for the first time. By subsequent Southern blot analyses, it could be demonstrated that during the integration of new spacers reaction intermediates are formed. A thorough analyses of these spacer integration intermediates revealed that Cas1 and Cas2 introduce staggered nicks specifically at the 5´-ends of the first repeat. Moreover, this staggered nicking is accompanied by the ligation of a new spacer DNA, suggesting a coupled cleavage-ligation (integrase) reaction performed by Cas1 and Cas2 proteins. In the second part of this work, the three Cas proteins Cse1, Cse2 and Cas7, which are constituents of the Cascade complex, were purified by affinity chromatography and analyzed for their ability to bind double- or single-stranded nucleic acids by mobility shift assays. The results showed a binding of the Cse1 protein to short double stranded and single-stranded DNA. Moreover, a specific binding of Cse1 to the pre-crRNA with high affinity could be demonstrated. In contrast, the Cse2 protein interacted selectively with single-stranded DNA. | |||||||
| Quelle: | Al-Attar, S., E. R. Westra, J. van der Oost and S. J. Brouns (2011). "Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes." Biol Chem 392(4): 277-289.
Arslan, Z., T. Stratmann, R. Wurm, R. Wagner, K. Schnetz and U. Pul (2013). "RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12." RNA Biol 10(5): 708-715. Arslan, Z., R. Wurm, O. Brener, P. Ellinger, L. Nagel-Steger, F. Oesterhelt, L. Schmitt, D. Willbold, R. Wagner, H. Gohlke, S. H. Smits and U. Pul (2013). "Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2." Nucleic Acids Res 41(12): 6347-6359. Arslan, Z., Hermanns, V., Wurm, R., Wagner, R., Pul, Ü., (2014). "Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system." Nucleic Acids Res (in Revision) Babu, M., N. Beloglazova, R. Flick, C. Graham, T. Skarina, B. Nocek, A. Gagarinova, O. Pogoutse, G. Brown, A. Binkowski, S. Phanse, A. Joachimiak, E. V. Koonin, A. Savchenko, A. Emili, J. Greenblatt, A. M. Edwards and A. F. Yakunin (2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair." Mol Microbiol 79(2): 484-502. Barrangou, R., C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D. A. Romero and P. Horvath (2007). "CRISPR provides acquired resistance against viruses in prokaryotes." Science 315(5819): 1709-1712. Barrangou, R. and P. Horvath (2012). "CRISPR: new horizons in phage resistance and strain identification." Annu Rev Food Sci Technol 3: 143-162. Beidler, J. L., P. R. Hilliard and R. L. Rill (1982). "Ultrasensitive staining of nucleic acids with silver." Anal Biochem 126(2): 374-380. Beloglazova, N., G. Brown, M. D. Zimmerman, M. Proudfoot, K. S. Makarova, M. Kudritska, S. Kochinyan, S. Wang, M. Chruszcz, W. Minor, E. V. Koonin, A. M. Edwards, A. Savchenko and A. F. Yakunin (2008). "A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats." J Biol Chem 283(29): 20361-20371. Bhaya, D., M. Davison and R. Barrangou (2011). "CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation." Annu Rev Genet 45: 273-297. Bolotin, A., B. Quinquis, A. Sorokin and S. D. Ehrlich (2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin." Microbiology 151(Pt 8): 2551-2561. Brouns, S. J., M. M. Jore, M. Lundgren, E. R. Westra, R. J. Slijkhuis, A. P. Snijders, M. J. Dickman, K. S. Makarova, E. V. Koonin and J. van der Oost (2008). "Small CRISPR RNAs guide antiviral defense in prokaryotes." Science 321(5891): 960-964. Burgess, R. R. and J. J. Jendrisak (1975). "A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography." Biochemistry 14(21): 4634-4638. Carte, J., R. Wang, H. Li, R. M. Terns and M. P. Terns (2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes." Genes Dev 22(24): 3489-3496. Chamberlin, M. J., W. C. Nierman, J. Wiggs and N. Neff (1979). "A quantitative assay for bacterial RNA polymerases." Journal of Biological Chemistry 254(20): 10061-10069. Chang, N., C. Sun, L. Gao, D. Zhu, X. Xu, X. Zhu, J. W. Xiong and J. J. Xi (2013). "Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos." Cell Res 23(4): 465-472. Charpentier, E. and J. A. Doudna (2013). "Biotechnology: Rewriting a genome." Nature 495(7439): 50-51. Cong, L., F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. A. Marraffini and F. Zhang (2013). "Multiplex genome engineering using CRISPR/Cas systems." Science 339(6121): 819-823. Cote, A. G. and S. M. Lewis (2008). "Mus81-dependent double-strand DNA breaks at in vivo-generated cruciform structures in S. cerevisiae." Mol Cell 31(6): 800-812. Datsenko, K. A., K. Pougach, A. Tikhonov, B. L. Wanner, K. Severinov and E. Semenova (2012). "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system." Nat Commun 3: 945. Deltcheva, E., K. Chylinski, C. M. Sharma, K. Gonzales, Y. Chao, Z. A. Pirzada, M. R. Eckert, J. Vogel and E. Charpentier (2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471(7340): 602-607. Deveau, H., R. Barrangou, J. E. Garneau, J. Labonte, C. Fremaux, P. Boyaval, D. A. Romero, P. Horvath and S. Moineau (2008). "Phage response to CRISPR-encoded resistance in Streptococcus thermophilus." J Bacteriol 190(4): 1390-1400. Diez-Villasenor, C., N. M. Guzman, C. Almendros, J. Garcia-Martinez and F. J. Mojica (2013). "CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli." RNA Biol 10(5): 792-802. Edgar, R. and U. Qimron (2010). "The Escherichia coli CRISPR system protects from lambda lysogenization, lysogens, and prophage induction." J Bacteriol 192(23): 6291-6294. Ellinger, P., Z. Arslan, R. Wurm, B. Tschapek, C. MacKenzie, K. Pfeffer, S. Panjikar, R. Wagner, L. Schmitt, H. Gohlke, U. Pul and S. H. Smits (2012). "The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae." J Struct Biol 178(3): 350-362. Gasiunas, G., R. Barrangou, P. Horvath and V. Siksnys (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria." Proc Natl Acad Sci U S A 109(39): E2579-2586. Gasiunas, G. and V. Siksnys (2013). "RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?" Trends Microbiol 21(11): 562-567. Gonzalez, N., J. Wiggs and M. J. Chamberlin (1977). "A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase." Arch Biochem Biophys 182(2): 404-408. Goren, M., I. Yosef, R. Edgar and U. Qimron (2012). "The bacterial CRISPR/Cas system as analog of the mammalian adaptive immune system." RNA Biol 9(5): 549-554. Goren, M. G., I. Yosef, O. Auster and U. Qimron (2012). "Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli." J Mol Biol 423(1): 14-16. Haft, D. H., J. Selengut, E. F. Mongodin and K. E. Nelson (2005). "A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes." PLoS Comput Biol 1(6): e60. Hale, C. R., P. Zhao, S. Olson, M. O. Duff, B. R. Graveley, L. Wells, R. M. Terns and M. P. Terns (2009). "RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex." Cell 139(5): 945-956. Hanahan, D. (1985). Techniques for transformation of E. coli. DNA cloning: a practical approach. D. M. Glover. Oxford, United Kingdom, IRL Press. 1: pp 109-135. Hatoum-Aslan, A., I. Maniv and L. A. Marraffini (2011). "Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site." Proc Natl Acad Sci U S A 108(52): 21218-21222. Haurwitz, R. E., M. Jinek, B. Wiedenheft, K. Zhou and J. A. Doudna (2010). "Sequence- and structure-specific RNA processing by a CRISPR endonuclease." Science 329(5997): 1355-1358. Hochstrasser, M. L., D. W. Taylor, P. Bhat, C. K. Guegler, S. H. Sternberg, E. Nogales and J. A. Doudna (2014). "CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference." Proc Natl Acad Sci U S A. Horvath, P. and R. Barrangou (2013). "RNA-guided genome editing a la carte." Cell Res 23(6): 733-734. Horvath, P., D. A. Romero, A. C. Coute-Monvoisin, M. Richards, H. Deveau, S. Moineau, P. Boyaval, C. Fremaux and R. Barrangou (2008). "Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus." J Bacteriol 190(4): 1401-1412. Ishino, Y., H. Shinagawa, K. Makino, M. Amemura and A. Nakata (1987). "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product." J Bacteriol 169(12): 5429-5433. Jansen, R., J. D. Embden, W. Gaastra and L. M. Schouls (2002). "Identification of genes that are associated with DNA repeats in prokaryotes." Mol Microbiol 43(6): 1565-1575. Jaskiewicz, L. and W. Filipowicz (2008). "Role of Dicer in posttranscriptional RNA silencing." Curr Top Microbiol Immunol 320: 77-97. Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna and E. Charpentier (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337(6096): 816-821. Jinek, M., A. East, A. Cheng, S. Lin, E. Ma and J. Doudna (2013). "RNA-programmed genome editing in human cells." Elife 2: e00471. Jore, M. (2010). CRISPR-mediated Antiviral Defence in Prokaryotes, publisher not identified. Jore, M. M., M. Lundgren, E. van Duijn, J. B. Bultema, E. R. Westra, S. P. Waghmare, B. Wiedenheft, U. Pul, R. Wurm, R. Wagner, M. R. Beijer, A. Barendregt, K. Zhou, A. P. Snijders, M. J. Dickman, J. A. Doudna, E. J. Boekema, A. J. Heck, J. van der Oost and S. J. Brouns (2011). "Structural basis for CRISPR RNA-guided DNA recognition by Cascade." Nat Struct Mol Biol 18(5): 529-536. Kunin, V., R. Sorek and P. Hugenholtz (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats." Genome Biol 8(4): R61. Larson, M. H., L. A. Gilbert, X. Wang, W. A. Lim, J. S. Weissman and L. S. Qi (2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression." Nat. Protocols 8(11): 2180-2196. Lemak, S., N. Beloglazova, B. Nocek, T. Skarina, R. Flick, G. Brown, A. Popovic, A. Joachimiak, A. Savchenko and A. F. Yakunin (2013). "Toroidal Structure and DNA Cleavage by the CRISPR-Associated [4Fe-4S] Cluster Containing Cas4 Nuclease SSO0001 from Sulfolobus solfataricus." Journal of the American Chemical Society 135(46): 17476-17487. Li, M., R. Wang, D. Zhao and H. Xiang (2014). "Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process." Nucleic Acids Res 42(4): 2483-2492. Lillestol, R. K., P. Redder, R. A. Garrett and K. Brugger (2006). "A putative viral defence mechanism in archaeal cells." Archaea 2(1): 59-72. Makarova, K. S., N. V. Grishin, S. A. Shabalina, Y. I. Wolf and E. V. Koonin (2006). "A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action." Biol Direct 1: 7. Makarova, K. S., D. H. Haft, R. Barrangou, S. J. Brouns, E. Charpentier, P. Horvath, S. Moineau, F. J. Mojica, Y. I. Wolf, A. F. Yakunin, J. van der Oost and E. V. Koonin (2011). "Evolution and classification of the CRISPR-Cas systems." Nat Rev Microbiol 9(6): 467-477. Marchfelder, A., L.-K. Maier, N. Heidrich and Ü. Pul (2013). "CRISPR-Cas." Biologie in unserer Zeit 43(3): 158-165. Marraffini, L. A. and E. J. Sontheimer (2008). "CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA." Science 322(5909): 1843-1845. Merril, C. R., D. Scholl and S. L. Adhya (2003). "The prospect for bacteriophage therapy in Western medicine." Nat Rev Drug Discov 2(6): 489-497. Mojica, F. J., C. Diez-Villasenor, J. Garcia-Martinez and C. Almendros (2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system." Microbiology 155(Pt 3): 733-740. Mojica, F. J., C. Diez-Villasenor, J. Garcia-Martinez and E. Soria (2005). "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements." J Mol Evol 60(2): 174-182. Mojica, F. M. and R. Garrett (2013). Discovery and Seminal Developments in the CRISPR Field. CRISPR-Cas Systems. R. Barrangou and J. van der Oost, Springer Berlin Heidelberg: 1-31. Mulepati, S., A. Orr and S. Bailey (2012). "Crystal structure of the largest subunit of a bacterial RNA-guided immune complex and its role in DNA target binding." J Biol Chem 287(27): 22445-22449. Mullis, K. B. and F. A. Faloona (1987). "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction." Methods Enzymol 155: 335-350. Nam, K. H., Q. Huang and A. Ke (2012). "Nucleic acid binding surface and dimer interface revealed by CRISPR-associated CasB protein structures." FEBS Lett 586(22): 3956-3961. Nam, K. H., I. Kurinov and A. Ke (2011). "Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity." J Biol Chem 286(35): 30759-30768. Nuñez, J. K., P. J. Kranzusch, J. Noeske, A. V. Wright, C. W. Davies and J. A. Doudna (2014). "Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity." Nat Struct Mol Biol advance online publication. Plagens, A., B. Tjaden, A. Hagemann, L. Randau and R. Hensel (2012). "Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax." J Bacteriol 194(10): 2491-2500. Popowitsch, A. (2013). "Bestimmung von kritischen CRISPR Leader-DNA-Sequenzen für die Immunisierung gegen Fremd-DNA" Bachelorarbeit, Heinrich-Heine-Universität. Pougach, K., E. Semenova, E. Bogdanova, K. A. Datsenko, M. Djordjevic, B. L. Wanner and K. Severinov (2010). "Transcription, processing and function of CRISPR cassettes in Escherichia coli." Mol Microbiol 77(6): 1367-1379. Pul, U., R. Wurm, Z. Arslan, R. Geissen, N. Hofmann and R. Wagner (2010). "Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS." Mol Microbiol 75(6): 1495-1512. Quax, Tessa E. F., Yuri I. Wolf, Jasper J. Koehorst, O. Wurtzel, R. van der Oost, W. Ran, F. Blombach, Kira S. Makarova, Stan J. J. Brouns, Anthony C. Forster, E. Gerhart H. Wagner, R. Sorek, Eugene V. Koonin and J. van der Oost (2013). "Differential Translation Tunes Uneven Production of Operon-Encoded Proteins." Cell Reports 4(5): 938-944. Sashital, D. G., B. Wiedenheft and J. A. Doudna (2012). "Mechanism of foreign DNA selection in a bacterial adaptive immune system." Mol Cell 46(5): 606-615. Schwabroch, T. (2012). "Charakterisierung von CRISPR/Cas Proteinen und die Rolle der CRISPR Leader-DNA bei der bakteriellen Immunisierung gegen Fremd-DNA" Masterarbeit, Heinrich-Heine-Universität. Semenova, E., M. M. Jore, K. A. Datsenko, A. Semenova, E. R. Westra, B. Wanner, J. van der Oost, S. J. Brouns and K. Severinov (2011). "Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence." Proc Natl Acad Sci U S A 108(25): 10098-10103. Shah, S. A., N. R. Hansen and R. A. Garrett (2009). "Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism." Biochem Soc Trans 37(Pt 1): 23-28. Sorek, R., V. Kunin and P. Hugenholtz (2008). "CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea." Nat Rev Microbiol 6(3): 181-186. Spreiz, V. (2011). "Klonierung und Charakterisierung der CRISPR-Gene in E. coli BL21", Heinrich-Heine-Universität. Stern, S., D. Moazed and H. F. Noller (1988). "Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension." Methods Enzymol 164: 481-489. Stratmann, T., U. Pul, R. Wurm, R. Wagner and K. Schnetz (2012). "RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants." Mol Microbiol 83(6): 1109-1123. Swarts, D. C., C. Mosterd, M. W. van Passel and S. J. Brouns (2012). "CRISPR interference directs strand specific spacer acquisition." PLoS One 7(4): e35888. Tang, T. H., J. P. Bachellerie, T. Rozhdestvensky, M. L. Bortolin, H. Huber, M. Drungowski, T. Elge, J. Brosius and A. Huttenhofer (2002). "Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus." Proc Natl Acad Sci U S A 99(11): 7536-7541. Tang, T. H., N. Polacek, M. Zywicki, H. Huber, K. Brugger, R. Garrett, J. P. Bachellerie and A. Huttenhofer (2005). "Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus." Mol Microbiol 55(2): 469-481. Terns, R. M. and M. P. Terns (2014). "CRISPR-based technologies: prokaryotic defense weapons repurposed." Trends Genet 30(3): 111-118. van der Oost, J., M. M. Jore, E. R. Westra, M. Lundgren and S. J. Brouns (2009). "CRISPR-based adaptive and heritable immunity in prokaryotes." Trends Biochem Sci 34(8): 401-407. Wagner, R. and Ü. Pul (2012). CRISPR: A Bacterial Immunity System Based on Small RNAs. From Nucleic Acids Sequences to Molecular Medicine. V. A. Erdmann and J. Barciszewski, Springer Berlin Heidelberg: 121-143. Westra, E. R., B. Nilges, P. B. van Erp, J. van der Oost, R. T. Dame and S. J. Brouns (2012). "Cascade-mediated binding and bending of negatively supercoiled DNA." RNA Biol 9(9): 1134-1138. Westra, E. R., U. Pul, N. Heidrich, M. M. Jore, M. Lundgren, T. Stratmann, R. Wurm, A. Raine, M. Mescher, L. Van Heereveld, M. Mastop, E. G. Wagner, K. Schnetz, J. Van Der Oost, R. Wagner and S. J. Brouns (2010). "H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO." Mol Microbiol 77(6): 1380-1393. Westra, E. R., E. Semenova, K. A. Datsenko, R. N. Jackson, B. Wiedenheft, K. Severinov and S. J. Brouns (2013). "Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition." PLoS Genet 9(9): e1003742. Westra, E. R., D. C. Swarts, R. H. Staals, M. M. Jore, S. J. Brouns and J. van der Oost (2012). "The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity." Annu Rev Genet 46: 311-339. Westra, E. R., P. B. van Erp, T. Kunne, S. P. Wong, R. H. Staals, C. L. Seegers, S. Bollen, M. M. Jore, E. Semenova, K. Severinov, W. M. de Vos, R. T. Dame, R. de Vries, S. J. Brouns and J. van der Oost (2012). "CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3." Mol Cell 46(5): 595-605. Wiedenheft, B., G. C. Lander, K. Zhou, M. M. Jore, S. J. J. Brouns, J. van der Oost, J. A. Doudna and E. Nogales (2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system." Nature 477(7365): 486-489. Wiedenheft, B., E. van Duijn, J. B. Bultema, S. P. Waghmare, K. Zhou, A. Barendregt, W. Westphal, A. J. Heck, E. J. Boekema, M. J. Dickman and J. A. Doudna (2011). "RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions." Proc Natl Acad Sci U S A 108(25): 10092-10097. Wiedenheft, B., K. Zhou, M. Jinek, S. M. Coyle, W. Ma and J. A. Doudna (2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense." Structure 17(6): 904-912. Woestenenk, E. A., M. Hammarstrom, S. van den Berg, T. Hard and H. Berglund (2004). "His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors." J Struct Funct Genomics 5(3): 217-229. Yosef, I., M. G. Goren and U. Qimron (2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli." Nucleic Acids Res 40(12): 5569-5576. Yosef, I., D. Shitrit, M. G. Goren, D. Burstein, T. Pupko and U. Qimron (2013). "DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array." Proc Natl Acad Sci U S A 110(35): 14396-14401. Zhang, J., T. Kasciukovic and M. F. White (2012). "The CRISPR associated protein Cas4 Is a 5' to 3' DNA exonuclease with an iron-sulfur cluster." PLoS One 7(10): e47232. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Molekularbiologie der Prokaryoten | |||||||
| Dokument erstellt am: | 11.07.2014 | |||||||
| Dateien geändert am: | 11.07.2014 | |||||||
| Promotionsantrag am: | 12.05.2014 | |||||||
| Datum der Promotion: | 18.06.2014 |
