Mitochondrial DNA helicases in the social amoeba Dictyostelium discoideum

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Physiology, Anatomy and Microbiology, School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

Thesis with publications.

Mitochondria contain their own genome which must be maintained for proper cellular function; this includes its replication and repair. This thesis explores mitochondrial DNA (mtDNA) helicases in Dictyostelium discoideum, an amoeba and model for mitochondrial genetics. Firstly Twm1, a replicative mtDNA helicase and Dictyostelium homologue of mammalian Twinkle, was identified and characterised, demonstrating that Twm1 unwinds DNA for replication and, unlike human Twinkle, possesses primase functionality. Twm1 was also found to recognise a sequence containing a likely origin of mtDNA replication, suggesting its involvement in initiating replication. It is important that mtDNA is properly maintained should it become damaged, processes in which Pif1 helicases are believed to play important roles. In this thesis, members of the Pif1 family in amoebae have been identified. While most organisms possess one or two Pif1 homologues, this study demonstrated that amoebae typically possess between two and nine; the D. discoideum genome encodes three. This work also identified the first collection of Pif1 helicases that contain additional domains, which might provide novel functionality for amoebal Pif1 proteins. Phylogenetic analysis suggested these domains were acquired through various genetic events, including horizontal gene transfer from bacteria and fungi. Finally, a candidate Pif1 homologue from D. discoideum (DdPif1) was experimentally characterised to understand its role in mtDNA maintenance. The results suggest that DdPif1 is involved in mtDNA recovery from both oxidative damage and fragmentation. Furthermore, DdPif1 was shown to be an active helicase important for resolving G-quadruplex DNA structures, which are predicted to form throughout the mitochondrial genome. Conversely, Twm1 was shown to be incapable of unwinding G-quadruplex containing DNA. This work has highlighted the significance of using Dictyostelium as a model for mitochondrial genetics, and has contributed to the understanding of mtDNA maintenance outside mammalian systems, mtDNA helicase functionality and their roles in maintaining the mitochondrial genome.