| dc.contributor.advisor | Jakobs, Stefan Prof. Dr. | |
| dc.contributor.author | Brüser, Christian | |
| dc.date.accessioned | 2019-03-13T09:23:06Z | |
| dc.date.available | 2019-03-13T09:23:06Z | |
| dc.date.issued | 2019-03-13 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-002E-E5C7-E | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7315 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Analysis of mitochondrial transcription and replication on the single nucleoid level | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Rehling, Peter Prof. Dr. | |
| dc.date.examination | 2018-05-17 | |
| dc.description.abstracteng | Human mitochondrial DNA (mtDNA) is a circular molecule encoding for 13 subunits of the respiratory chain that are essential for its maintenance. Inside the mitochondria, mtDNA is associated with various proteins, forming a nucleoprotein complex termed a nucleoid. Mutations of the mtDNA as well as deviations in the composition of the associated proteins are implicated in a variety of diseases in humans. However, the regulation of the basic nucleoid functions, i.e. replication and transcription on the single nucleoid level is poorly understood.
Human cells contain up to several hundreds of nucleoids, each with an average diameter of about 80 nm. Since nucleoids cluster together within mitochondria, the resolution and analysis of single nucleoids is impaired in diffraction-limited microscopy methods. In this work, nanoscopy is used to visualize single nucleoids and to address fundamental questions about the transcription and replication of mtDNA.
For this purpose, a robust staining protocol to identify nucleoids engaged in replication or transcription, was established based on the incorporation of synthetic nucleosides. Three-color STED nanoscopy revealed the organization of nucleoids into at least two subpopulations within single cells. The results of this study indicate that nucleoids can either be active with a tendency to be engaged in both processes or seemingly totally inactive. This finding provides new explanations for the high copy number of the mitochondrial genome.
In addition, the approach developed here was used to analyze knockdown cells of important key players of mitochondrial regulation, namely, the mitochondrial RNA Polymerase POLRMT and mitochondrial elongation factor TEFM. POLRMT is required for functional transcription and synthesis of the replication primer, while the switch between mitochondrial transcription and replication is mediated by TEFM.
The present work demonstrates that POLRMT and TEFM together coordinate whether the mtDNA is used as a template for transcription or replication. Upon POLRMT reduction, transcription is favored over replication of mtDNA, whereas a decrease in the expression of TEFM leads to increased replication. This provides fundamental insights into the reciprocal regulation of mitochondrial transcription and replication. | de |
| dc.contributor.coReferee | Mansouri, Ahmed Prof. Dr. | |
| dc.subject.eng | Mitochondria | de |
| dc.subject.eng | mtDNA | de |
| dc.subject.eng | POLRMT | de |
| dc.subject.eng | TEFM | de |
| dc.subject.eng | high-resolution microscopy | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E5C7-E-7 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1673693210 | |