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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Michele M Klingbeil
Steven J. Sandler
Yasu S. Morita
Kinetoplastid organisms including medically relevant species like Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy number and sequence classes among different species, kDNA is most often found as a network of thousands of DNA molecules catenated together. This unique biological property of disease-causing organisms has been a subject of study as a potential drug target since it is essential for parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and related diseases in other mammals and is fatal if left untreated. Recent advances in drug development have produced an oral medication to treat most forms of the disease, hopefully alleviating the medical burden of the organism and enabling us to appreciate and study its fascinating mitochondrial biology.
Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. In Chapter 2, we show that POLIC localization changes throughout kDNA S phase and in order to look for potential interactions with kDNA segregation machinery, we utilize structured illumination microscopy to follow localization dynamics with a TAC marker, TAC102. Additionally, we show that kDNA replication proteins POLIB and POLID do not affect TAC102 localization further reinforcing them as strictly kDNA replication proteins. In Chapter 3, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.
Miller, Jonathan, "STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE" (2020). Doctoral Dissertations. 1938.
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