Specialized Mitochondrial DNA Polymerases in African Trypanosomes

Trypanosomatid parasites infect diverse species of insects, animals, and plants, but are best known as causative agents of the Neglected Tropical Diseases human African trypanosomiasis, Chagas disease and Leishmaniasis, some of which are fatal without intervention. New, selective, short-course trypanocidals are needed for human and animal infections. Trypanosomes have one of the most complex mitochondrial genomes in nature called kinetoplast DNA (kDNA) that is a catenated network composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent from all other eukaryotes, and is essential for parasite survival and life cycle completion. Prominent features of the kDNA replication model include release and attachment of minicircles, and remarkably, persistent nicks and gaps in the newly replicated molecules that are not repaired until the segregation of daughter networks. This suggests that the DNA damage tolerance response during kDNA replication is essential and therefore notably different in trypanosomatids. Lastly, while most eukaryotes rely on one essential mitochondrial DNA polymerase (Pol), T. brucei uses three family A DNA Pol paralogs (POLIB, POLIC, POLID) that are independently essential to maintain the kDNA network suggesting these proteins have evolved specialized functions. Our long-term goal of determining how multiple DNA Pols are used to maintain the kDNA network is key to gaining a deeper understanding of how to selectively interfere with this essential process. This project will clarify the roles of POLIB and POLID in kDNA replication and evaluate essential functional domains responsible for responding to replication stress. We address the functions of POLIB and POLID using a system with multiple inducers that allow independent, tunable, and temporal regulation of gene expression for detailed mechanistic studies. Aim 1 will define roles of POLIB and POLID in kDNA replication through RNAi complementation with a focus on maxicircle replication and proofreading replicative roles. Aim 2 will establish a novel triple control induction system to induce kDNA-specific replication stress and subsequently evaluate the roles of POLIB and POLID under replication stress conditions through RNAi complementation. Our findings will clarify the specialized roles of POLIB and POLID paving the way to elucidate the division of labor among the Pol paralogs, a feature that has been enigmatic in the long-held kDNA replication model. The Pol paralogs are preserved across kinetoplastids, therefore a mechanistic understanding of POLIB and POLID in T. brucei has the potential for a broad-based anti-trypanocidal, and to provide a rare opportunity to study features that have evolved for Family A DNA Pols.