Trypanosomatid Mitochondrial DNA PolymerasesSummaryPrincipal Investigator: Michele Klingbeil Abstract: DESCRIPTION (provided by the applicant): 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. Funding Period: 2005-08-01 - 2010-01-31 more information: NIH RePORT Top Publications
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Publications
Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes
Julian Chandler
Department of Microbiology, University of Massachusetts, 639 North Pleasant Street, Amherst, MA 01003, USA
Eukaryot Cell 7:2141-6. 2008..While other eukaryotes utilize a single DNA Pol (Pol gamma) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network...Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes
David F Bruhn
Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA
Mol Microbiol 75:1414-25. 2010..Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery...Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei
Amy L Springer
Department of Biology, Amherst College, Amherst, MA, USA
Mol Biochem Parasitol 175:68-75. 2011....Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei
David F Bruhn
Department of Microbiology, University of Massachusetts, 639 North Pleasant Street, Amherst, MA 01003, USA
Eukaryot Cell 10:734-43. 2011....Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID
Jeniffer Concepción-Acevedo
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA
Eukaryot Cell 11:844-55. 2012..Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication...