: Trypanosoma brucei is a protist parasite that causes fatal African sleeping sickness in humans, and a related disease in cattle called nagana. Current drug treatments are inadequate, often toxic, and no vaccine is available. T. brucei and other trypanosomatids are distinguished from all other eukaryotes by an unusual mitochondrial genome that is a catenated network of maxicircles (tens) and minicircles (thousands) called kinetoplast DNA (kDNA). No replicative mitochondrial DNA polymerase (pol) has been identified from trypanosomatids. However, we have identified four T. brucei mitochondrial proteins (Tbpol IA, IB, IC and ID) related to bacterial pol I. This is unique because yeast and vertebrates utilize just a single mitochondrial replicative enzyme, pol gamma. Our long term goal is to understand the particular roles these multiple mitochondrial pols play in maintaining kDNA which is essential for parasite viability. The specific hypothesis is that multiple pol I-like proteins are the kDNA replicative enzymes. We base our hypothesis on the following observations: 1) pol IB and IC localize near the kDNA where minicircle replication initiates, 2) single gene RNA interference (RNAi) experiements indicate that both are required for normal growth of procyclic parasites and 3) silencing of IB or IC results in kDNA network shrinkage and an accumulation of minicircle replication intermediates, but not a complete block in minicircle replication. We will use a multifaceted approach including genetics, molecular biology, and biochemistry to identify replicative properties of the mitochondrial pol I-like proteins. Our three specific aims are focused on pol IB, IC and ID Aim 1. Clarify the cellular role(s) of the pol I-like proteins by using various RNAi cell lines including single, and multiple knockdown constructs.
Aim 2. Characterize the enzymatic properties of recombinant pol I-like proteins, including processivity, fidelity, and inhibitor studies.
Aim 3. Identify associated proteins that contribute to the specific pol cellular roles. Completion of these specific aims will provide important new information about kDNA replication, a process conserved in trypanosomatids, and possibly the opportunity to exploit differences between trypanosomatid and mammalian mitochondrial DNA pols to develop new strategies for drug treatment. The T. brucei pol I-like protein family also offers the unique opportunity to uncover insights into the evolutionary strategies used to produce a replicative polymerase.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI066279-02
Application #
7104395
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
2005-08-01
Project End
2009-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
2
Fiscal Year
2006
Total Cost
$306,214
Indirect Cost
Name
University of Massachusetts Amherst
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Concepcion-Acevedo, Jeniffer; Luo, Juemin; Klingbeil, Michele M (2012) Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID. Eukaryot Cell 11:844-55
Springer, Amy L; Bruhn, David F; Kinzel, Kathryn W et al. (2011) Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei. Mol Biochem Parasitol 175:68-75
Bruhn, David F; Sammartino, Mark P; Klingbeil, Michele M (2011) Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei. Eukaryot Cell 10:734-43
Bruhn, David F; Mozeleski, Brian; Falkin, Laurie et al. (2010) Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes. Mol Microbiol 75:1414-25
Chandler, Julian; Vandoros, Anthula V; Mozeleski, Brian et al. (2008) Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes. Eukaryot Cell 7:2141-6