The doctoral dissertation in the field of Biology will be examined at the Faculty of Science, Forestry and Technology, Joensuu campus and online.
What is the topic of your doctoral research? Why is it important to study the topic?
My doctoral research focuses on the mechanisms of mitochondrial DNA (mtDNA) replication initiation, with emphasis on the roles of transcription and recombination. Although mtDNA replication is essential for cellular energy production and regulates several cellular mechanisms, the processes that trigger replication initiation in mammalian mitochondria remain incompletely understood. Mitochondria can employ multiple replication modes, and how cells choose between them under normal and stress conditions is still under debate. Impaired mtDNA maintenance underlies mitochondrial diseases and leads to dysfunction in energy-demanding tissues such as brain, muscle, and heart.
By clarifying how transcription-derived RNA primers and recombination-dependent pathways support mtDNA replication, this research provides insight into how mitochondria preserve their genomes, especially when replication is challenged by stress.
What are the key findings or observations of your doctoral research?
This research shows that the initiation of mtDNA replication is flexible and depends on the cellular situation. Under normal conditions, priming of mtDNA replication relies on mitochondrial transcription. When transcription is partially reduced, short RNA fragments are still sufficient to start DNA replication.
However, when mitochondrial transcription is completely blocked, replication initiation fails, demonstrating a direct link between transcription activity and mtDNA replication. Reduced transcription also changes the structure of mtDNA, indicating that RNA has an additional structural role beyond simply acting as a replication primer.
The research further demonstrates that recombination can support mtDNA replication when cells experience replication stress. By combining several complementary methods, recombination structures and recombinant mtDNA molecules were identified in mammalian cells. Although the existence of mitochondrial recombination in mammals has been debated, these findings provide evidence that recombination, and more specifically recombination-dependent replication, operates in mammalian mitochondria, particularly under stress conditions.
These results show that mitochondria can switch between transcription-driven and recombination-mediated replication initiation pathways, which means that mtDNA maintenance is very flexible.
What are the key research methods and materials used in your doctoral research?
This study used multiple molecular biology and DNA analysis techniques to investigate mtDNA replication initiation under both normal and stress conditions, improving our understanding of mitochondrial genome stability. I used human and mouse cell lines and inhibitors to selectively interfere with mitochondrial transcription and replication.
mtDNA copy number, transcription levels, topology, and replication intermediates were analyzed using quantitative PCR, Southern blotting, and two-dimensional agarose gel electrophoresis. The structure of mtDNA and recombination intermediates were also examined by transmission electron microscopy.
High-resolution sequencing approaches were used to detect recombinant mtDNA molecules, including Nanopore and PacBio HiFi sequencing. Confocal immunofluorescence microscopy was used to assess mitochondrial organization and nucleoid structure.
The doctoral dissertation of Georgios Fragkoulis, MSc, entitled Mechanisms of mitochondrial DNA replication initiation: roles of transcription and recombination be examined at the Faculty of Science, Forestry and Technology, Joensuu campus. The opponent will be Academy Research Fellow, Docent Tiina S. Salminen, Tampere University, and the custos will be University Lecturer Steffi Goffart University of Eastern Finland. Language of the public defence is English.
