Trypanosomatid Mitochondrial DNA Polymerases

Summary

Principal 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

  1. pmc 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
  2. ncbi 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
  3. pmc 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
  4. pmc 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
  5. pmc 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

Detail Information

Publications5

  1. pmc 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...
  2. ncbi 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...
  3. pmc 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
    ....
  4. pmc 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
    ....
  5. pmc 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...