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

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.

Summary:
All functions of each cell are encoded in the genome, which is distributed equally to the daughter cells during each cell division. This applies to chromosomes, regardless the number of chromosomes. The integrity of the genome is essential for the survival of any organism. Using synthetic biology methods, extensive alterations of genomes or entire chromosomes can be synthesized, although the focus is mostly on the coding sequences. However, chromosomes are more than merely a sequential arrangement of genes. Chromosomes need systems for replication, segregation, organization, and repair, which is often done by interactions of proteins with DNA sequence motifs. The present study investigates such so-called chromosome maintenance systems using synthetic secondary chromosomes in E. coli. Can the results generate a better understanding of DNA replication in bacteria? The synthetic secondary chromosome (synVicII) established in this work replicates in E. coli similarly to the secondary chromosome in V. cholerae on which it is based. After an initial characterization, the design of synVicII was further optimized. A crucial step was to generate compatibility of synVicII with the hierarchical DNA assembly system MoClo. In parallel, an approach was established to generate highly variable, long DNA sequences in which user-defined DNA motifs can be excluded. The insertion of these DNA sequences into synVicII allowed the construction of synthetic secondary chromosomes with a size of 100 kb. These chromosomes make it feasible for the first time to analyze the interaction of DNA segregation and DNA mismatch repair in vivo by a set of three synthetic secondary chromosomes. Both processes are dependent on the presence of hemi-methylated GATC sequences in E. coli. This work shows that a differentiated binding of the two responsible proteins, for the above processes, SeqA and MutH, can be achieved by an ordered GATC arrangement to allow comparative analysis. Since the functionality of SeqA has not yet been fully understood, the quantitative understanding of SeqA was experimentally improved. Based on this data a model for SeqA structure variants at the replication fork was generated. It was demonstrated by FRAP experiments that SeqA is a dynamic protein that diffuses freely between two binding events within the cell. SeqA and Dam compete for the hemi-methylated GATCs. It was shown that both proteins are in a similar ratio to each other. This could be an aspect for how GATC re-methylation is regulated. The results of the present work contribute significantly to the understanding of DNA replication in bacteria.

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