Publikationsserver der Universitätsbibliothek Marburg
Titel:Konstruktion synthetischer sekundärer Chromosomen zur Charakterisierung von DNA-Reparatur und Segregation in Escherichia coli
Autor:Schindler, Daniel
Weitere Beteiligte: Waldminghaus, Torsten (Prof. Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0043
URN: urn:nbn:de:hebis:04-z2017-00430
DOI: https://doi.org/10.17192/z2017.0043
DDC: Biowissenschaften, Biologie
Titel (trans.):Construction of synthetic secondary chromosomes for characterization of DNA repair and segregation in Escherichia coli
Publikationsdatum:2017-01-12
Lizenz:https://creativecommons.org/licenses/by/4.0

Dokument

Schlagwörter:
synthetic biology, Vibrio cholerae, synthetic chromosomes, Escherichia coli, Vibrio cholerae, Escherichia coli, Synthetische Biologie, DNA repair, DNS-Reparatur, Replikation

Zusammenfassung:
Alle Funktionen einer jeden Zelle sind im Genom kodiert, dieses wird in jeder Zellteilung – egal ob ein oder mehrere Chromosomen – gleich auf die Tochterzellen verteilt. Die Integrität des Genoms ist für das Überleben eines jeden Organismus essentiell. Durch die Methoden der Synthetischen Biologie werden umfangreiche Veränderungen an Genomen durchgeführt bzw. ganze Chromosomen synthetisiert, wobei der Fokus meist auf den kodierenden Sequenzen liegt. Chromosomen sind aber mehr als eine Aneinanderreihung von Genen. Chromosomen benötigen Systeme zur Replikation, Segregation, Organisation und Reparatur, was häufig über Wechselwirkungen von Proteinen mit DNA-Sequenzmotiven geschieht. Die vorliegende Arbeit studiert solche, als Chromosome Maintenance System bezeichnete Prozesse anhand von synthetischen sekundären Chromosomen in E. coli. Können die resultierenden Ergebnisse zum Verständnis der DNA-Replikation in Bakterien beitragen? Das im Rahmen dieser Arbeit etablierte synthetische sekundäre Chromosom (synVicII) repliziert in E. coli wie das sekundäre Chromosom in V. cholerae, auf dem es basiert. Das Design wurde nach einer initialen Charakterisierung weiter optimiert. Ein entscheidender Schritt war die Herstellung einer Kompatibilität von synVicII mit dem hierarchischen DNA-Assemblierungssystem MoClo. Parallel wurde eine Vorgehensweise etabliert, um hochvariable, lange DNA-Sequenzen zu generieren, in denen benutzerdefinierte DNA-Sequenzen ausgeschlossen werden können. Die Insertion dieser DNA-Sequenzen in synVicII ermöglicht es, synthetische sekundäre Chromosomen mit einer Größe von 100 kb zu konstruieren. Dadurch konnte im Rahmen der vorliegenden Arbeit erstmals die Interaktion der DNA-Segregation und DNA mismatch Reparatur in vivo durch ein Set von drei synthetischen sekundären Chromosomen analysiert werden. Beide Prozesse sind auf das Vorhandensein hemi-methylierter GATC-Sequenzen angewiesen und die Arbeit zeigt, dass durch eine strukturierte GATC-Anordnung ein differenzielles Binden der beiden Proteine SeqA und MutH erreicht werden kann. Da die Funktionsweise von SeqA noch nicht vollständig verstanden ist, wurde außerdem das quantitative Verständnis von SeqA experimentell verbessert. Anhand der Daten wurde ein Modell der SeqA-Strukturen an den Replikationsgabeln generiert. Durch FRAP-Experimente konnte belegt werden, dass SeqA ein dynamisches Protein ist, welches zwischen zwei Bindeereignissen frei in der Zelle diffundiert. SeqA und Dam konkurrieren um die hemi-methylierten GATCs. Es konnte gezeigt werden, dass beide Proteine in einem konstanten Mengenverhältnis vorliegen. Dies könnte ein möglicher Aspekt zur Regulation der Re-methylierung der GATC-Sequenzen in E. coli sein. Die Ergebnisse der vorliegenden Arbeit tragen dadurch signifikant zum Verständnis der DNA-Replikation in Bakterien bei.

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