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The first part of this work focuses on the investigation of DNA translocases in the Gram positive model organism Bacillus subtilis, which move the chromosomes away from the division plane by directed DNA transport. SpoIIIE is a membrane-integral translocase that also acts during sporulation, while SftA is associated with the division septum by an unknown mechanism and was reported to be a soluble protein in vitro. The solubility of SftA in vivo was proven with cell fractionation experiments, and the part of the protein that serves for septal targeting was determined through the localization of different truncations of fluorescently labeled SftA, which helped narrow down the stretch of amino acids responsible for targeting the protein to the septum. Expression in a Eukaryotic heterologous system revealed an interaction between SftA and FtsA, but not FtsZ. Further evidence was provided by single molecule tracking experiments whereby the fraction of soluble SftA molecules increased in an FtsA depletion background as compared to a wild type background. Concomitant investigation on the single molecule level of SpoIIIE, a membrane associated DNA translocase in B. subtilis, and PfkA, a soluble phosphofructokinase, revealed a different behavior of the two translocases (SftA and SpoIIIE): SftA has a septal bound fraction, and a small soluble fast moving fraction,comparable in diffusion coefficient to the exclusively soluble PfkA tracked under the same conditions. SpoIIIE is much slower in comparison, and even its “fast” moving fraction is much slower than that of SftA. It seems to move slowly along the membrane with no specific enrichment at the septum, even after Mitomycin C (MMC) treatment. The second part of this work focused on the count of nucleoids, origin, and terminus of replication regions under conditions of fast and slow growth, with or without the induction of double strand breaks with MMC. B. subtilis seemed to be predominantly diploid, with a fraction of polyploid and monoploid cells which changed depending on the growth conditions. Replication of the origin proximal regions increased after DNA damage induction, as was shown by the increase in the number of origins during the time of DNA repair, while the number of termini remained constant. Time lapse experiments of the segregation of the tagged origin regions revealed that the movement is best described as directed diffusion, but seems to be quite robust and continues, though slower, after MMC treatment or treatment with Ciprofloxacin which blocks the topoisomerase IV 2017-09-14 2016 opus:7158 Life sciences Biowissenschaften, Biologie 2017-09-14 Philipps-Universität Marburg Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg 152 application/pdf DNA Translokalisation und Segregation von Ursprungsregionen in B. subtilis urn:nbn:de:hebis:04-z2017-00754 monograph Die Segregation von Chromosomen wird durch spezielle Mechanismen sicher gestellt, welche die Position des Septums sowie die korrekte Trennung von chromosomalen Dimeren gewährleisten und somit an der Aufrechterhaltung der Integrität der chromosomalen DNA während des gesamten Zellzyklus beteiligt sind. Anhand des Gram-positiven Modellorganismus Bacillus subtilis wurden im ersten Teil dieser Arbeit DNA-Translokasen untersucht, welche über einen direkten Transport die Chromosomen von der zellulären Teilungsebene weg bewegen. Bei SpoIIIE handelt es sich um eine Membranintegrierte Translokase, die während der Sporulation aktiv ist. Es wurde bereits gezeigt, dass SftA hingegen in vitro löslich ist und über einen bisher unbekannten Mechanismus mit dem Divisionsseptum assoziiert. Die Löslichkeit von SftA in vivo wurde über Zell-FraktionierungsExperimente nachgewiesen. Der Teil des Proteins, welcher zur Bindung an das Septum führt, wurde über die Lokalisation von unterschiedlichen, trunkierten und fluoreszenzmarkierten SftA Derivaten identifiziert, wodurch die für die Bindung an das Septum verantwortlichen Aminosäuren eingegrenzt werden konnten (siehe Manuskript, 4A). Über die Expression in einem eukaryotischen, heterologen System konnte eine Interaktion von SftA mit FtsA, jedoch nicht mit FtsZ nachgewiesen werden. Ein weiterer Nachweis hierfür wurde über Experimente durch single molecule tracking erbracht, wobei die lösliche Fraktion der SftA-Moleküle bei geringer Expression von FtsA im Vergleich zu dem Wildtyp zunimmt. Untersuchungen auf Einzelmolekül-Level von SpoIIIE, einer Membran-assoziierten DNATranslokase in B. subtilis und PfkA, einer löslichen Phosphofructokinase, ließen auf ein unterschiedliches Verhalten der beiden Translokasen (SftA und SpoIIIE) schließen: SftA besitzt eine am Septum gebundene Fraktion und eine kleine, sich schnell bewegende, lösliche Fraktion, welche mit der ausschließlich löslichen PfkA-Fraktion vergleichbar ist. SpoIIIE ist im Vergleich weniger dynamisch, wobei sogar die dynamische Fraktion viel langsamer ist als die statische SftA Fraktion. Scheinbar bewegt sich SpoIIIE langsam entlang der Membran, ohne sich am Septum anzureichern; selbst nach der Zugabe von Mitomycin C (MMC, siehe Manuskript 4B). Der zweite Teil dieser Arbeit befasste sich mit der Anzahl von Nukleoiden, Origin- und Termini unter schnellen und langsamen Wachstumsbedingungen, mit oder ohne Induktion von Doppelstrangbrüchen durch MMC. B. subtilis scheint vornehmlich diploid zu sein, mit polyploiden und monoploiden Zellfraktionen, welche sich abhängig von den gewählten Wachstumsbedinungen verändern. Die Replikation von benachbarten Regionen der Replikationsursprünge nahm nach der Induktion von DNA-Schäden zu, wie schon durch die Zunahme der Anzahl von Origins während der DNA-Reparatur gezeigt wurde, während die Anzahl der Terminatorsequenzen konstant blieb. Time lapse Experimente der Segregation von markierten Replikationsursprüngen zeigte, dass sich die Bewegung der Moleküle am besten durch gezielte Diffusion beschreiben lässt, welche sich durch die Zugabe von MMC oder Ciprofloxacin, welches die Topoisomerase IV inhibiert, zwar verlangsamt, sich jedoch als robust und kontinuierlich beschreiben lässt doctoralThesis English El Najjar, Nina El Najjar Nina https://doi.org/10.17192/z2017.0075 https://archiv.ub.uni-marburg.de/diss/z2017/0075/cover.png B. subtilis ths Prof. Dr. Graumann Peter Graumann, Peter (Prof. Dr.) 2016-12-14 DNA translocases and origin region segregation in B. subtilis Segregation