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Charakterisierung Des Replikationsenzyms ORF904 Aus Sulfolobus Islandicus

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Charakterisierung Des Replikationsenzyms ORF904 Aus Sulfolobus Islandicus Charakterisierung des Replikationsenzyms ORF904 aus Sulfolobus islandicus DISSERTATION zur Erlangung des Grades - Doktor der Naturwissenschaften - der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Diplom-Biologe Martin Sanchez Bayreuth 2009 Die vorliegende Arbeit wurde von September 2005 bis September 2009 am Lehrstuhl für Biochemie der Universität Bayreuth unter Anleitung von Prof. Dr. Georg Lipps angefertigt. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.). Promotionsgesuch eingereicht am: 22.09.2009 Tag des wissenschaftlichen Kolloquiums: 18.01.2010 Prüfungsausschuss: Prof. Dr. Georg Lipps (Erstgutachter) Prof. Dr. Wolfgang Schumann (Zweitgutachter) Prof. Dr. Carlo Unverzagt (Vorsitzender) Prof. Dr. Birgitta Wöhrl Meinen Eltern Inhaltsverzeichnis I Inhaltsverzeichnis 1 Einleitung ..............................................................................................................................................1 1.1 Die Replikationsinitiation..................................................................................................................1 1.2 AAA+-ATPasen.................................................................................................................................4 1.3 Helikasen .........................................................................................................................................7 1.4 SF3-Helikasen..................................................................................................................................9 1.5 Archaeale Plasmide ...................................................................................................................... 12 1.6 Das archaeale Plasmid pRN1....................................................................................................... 12 1.7 Die Replikationsinitiation von pRN1.............................................................................................. 14 1.8 Problemstellung ............................................................................................................................ 15 2 Material und Methoden ...................................................................................................................... 16 2.1 Materialien..................................................................................................................................... 16 2.2 Puffer, Lösungen und Medien....................................................................................................... 24 2.2.1 Bakteriennährmedien............................................................................................................ 24 2.2.1.1 Medienzusätze ........................................................................................................... 24 2.2.1.1.1 Agar .............................................................................................................. 24 2.2.1.1.2 Antibiotika..................................................................................................... 24 2.2.2 Puffer für die Herstellung chemisch kompetenter E. coli-Zellen........................................... 25 2.2.3 Lösung für die Gewinnung einzelsträngiger DNA aus Phagen............................................. 25 2.2.4 Puffer für die Phenol/Chloroformextraktion........................................................................... 25 2.2.5 Puffer für die Polymerasekettenreaktion (Polymerase Chain Reaction, PCR)..................... 25 2.2.6 Puffer für enzymatische Reaktionen mit DNA....................................................................... 26 2.2.7 Puffer für die Agarosegelelektrophorese .............................................................................. 26 2.2.8 Puffer für die Polyacrylamidgelelektrophorese (PAGE)........................................................ 26 2.2.8.1 Denaturierende PAGE ............................................................................................... 26 2.2.8.2 Native PAGE .............................................................................................................. 26 2.2.8.3 Diskontinuierliche SDS-PAGE nach Schägger und von Jagow................................. 27 2.2.9 Puffer für den Westernblot .................................................................................................... 27 2.2.10 Puffer für die Chromatographie........................................................................................... 27 2.2.10.1 Talon ........................................................................................................................ 27 - 2.2.10.2 EMD SO3 /EMD DEAE/EMD TMAE......................................................................... 28 2.2.10.3 Hydroxylapatit .......................................................................................................... 28 2.2.10.4 EMD Propyl .............................................................................................................. 28 2.2.10.5 Gelfiltration (TSK-gel G3000SW)............................................................................. 28 2.2.11 Puffer für die Dialyse von Proteinen ................................................................................... 28 2.2.12 Puffer für die Untersuchung der Proteinaktivitäten ............................................................. 29 2.2.12.1 ATPase-, Translokations- und Entwindungsaktivität ............................................... 29 2.2.12.2 Triple-Helix-Entwindung........................................................................................... 29 2.2.12.3 dsPlasmid-Entwindung............................................................................................. 29 2.2.12.4 Fluoreszenzanisotropie............................................................................................ 29 Inhaltsverzeichnis II 2.2.12.5 CD-Spektroskopie .................................................................................................... 29 2.2.12.6 Limitierende Proteolyse............................................................................................ 30 2.2.12.7 Glyceringradientenzentrifugation ............................................................................. 30 2.2.12.8 DNase-I-Nuklease-Footprint .................................................................................... 30 2.2.12.9 KMnO4-Footprint ...................................................................................................... 30 2.2.13 Größenstandards für die Gelelektrophorese....................................................................... 31 2.2.13.1 DNA-Standards ........................................................................................................ 31 2.2.13.2 Proteinstandards ...................................................................................................... 31 2.3 Methoden ...................................................................................................................................... 32 2.3.1 Anzucht von Bakterien .......................................................................................................... 32 2.3.2 Herstellung kompetenter E. coli und Transformation............................................................ 32 2.3.2.1 Rubidiumchloridmethode zur Herstellung kompetenter E. coli.................................. 32 2.3.2.2 Transformation von E. coli ......................................................................................... 32 2.3.3 Präparation und Nachweis von Nukleinsäuren..................................................................... 32 2.3.3.1 Schnellpräparation von Plasmid-DNA........................................................................ 32 2.3.3.2 Mini- und Midipräparation von Plasmid-DNA............................................................. 33 2.3.3.3 Präparation einzelsträngiger Plasmid-DNA ............................................................... 33 2.3.3.4 Phenol/Chloroformextraktion ..................................................................................... 34 2.3.3.5 Ethanolpräzipitation.................................................................................................... 34 2.3.3.6 Bestimmung der Konzentration von DNA .................................................................. 34 2.3.4 Klonierung von DNA-Fragmenten......................................................................................... 34 2.3.4.1 PCR............................................................................................................................ 34 2.3.4.2 Verwendung von Klonierungsvektoren ...................................................................... 35 2.3.4.2.1 pGEMT ......................................................................................................... 35 2.3.4.2.2 pJET1.2/blunt ............................................................................................... 36 2.3.4.3 Quickchange-Mutagenese ......................................................................................... 36 2.3.4.4 Sequenzierung von DNA-Fragmenten....................................................................... 36 2.3.5 Enzymatische Reaktionen mit DNA .....................................................................................
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