Cell Cycle Localization of DNA Polymerase IB in African trypanosomes

Trypanosoma brucei is a single celled eukaryote that causes the neglected tropical disease Human African Trypanosomiasis (HAT) in humans and a wasting disease called Nagana in cattle. An interesting feature is its unusual mitochondrial DNA network called the kinetoplast DNA (kDNA). Faithful replication of the kDNA network is essential for parasite survival and requires a multiplicity of proteins. While most eukaryotes use one mitochondrial DNA polymerase (Pol γ), T. brucei uses three DNA polymerase paralogs (POLIB, IC and ID), all of which have non-redundant roles in kDNA replication. We are studying the mechanisms that govern the choreography of kDNA replication localization dynamics. We demonstrated that POLIC and POLID display spatiotemporal localization and transiently associate to discrete protein assemblies flanking the kDNA disk during kDNA replication. This project is focused on defining the precise localization and dynamics of the third essential paralog, POLIB.

Techniques will include using molecular cloning techniques such as PCR cloning and Gibson assembly, to generate allelically tagged transgenic cell lines with various epitope tags (HA, Flag, Ty1). The student will also learn basic cell culturing techniques, nucleofection, western blotting and immunofluorescence microscopy using the Light Microscopy Core Facility located within the Institute of Applied Life Sciences. We are looking for someone who can minimally stay for a year, but ideally longer. Prior experience with molecular cloning will be helpful.