@article {2372,
	title = {Cell cycle localization dynamics of mitochondrial DNA polymerase IC in African trypanosomes.},
	journal = {Mol Biol Cell},
	volume = {29},
	year = {2018},
	month = {10/2018},
	pages = {2540-2552},
	abstract = {<p>Trypanosoma brucei has a unique catenated mitochondrial DNA (mtDNA) network called kinetoplast DNA (kDNA). Replication of kDNA occurs once per cell cycle in near synchrony with nuclear S phase and requires the coordination of many proteins. Among these are three essential DNA polymerases (TbPOLIB, IC, and ID). Localization dynamics of these proteins with respect to kDNA replication stages and how they coordinate their functions during replication are not well understood. We previously demonstrated that TbPOLID undergoes dynamic localization changes that are coupled to kDNA replication events. Here, we report the localization of TbPOLIC, a second essential DNA polymerase, and demonstrate the accumulation of TbPOLIC foci at active kDNA replication sites (antipodal sites) during stage II of the kDNA duplication cycle. While TbPOLIC was undetectable by immunofluorescence during other cell cycle stages, steady-state protein levels measured by Western blot remained constant. TbPOLIC foci colocalized with the fraction of TbPOLID that localized to the antipodal sites. However, the partial colocalization of the two essential DNA polymerases suggests a highly dynamic environment at the antipodal sites to coordinate the trafficking of replication proteins during kDNA synthesis. These data indicate that cell cycle-dependent localization is a major regulatory mechanism for essential mtDNA polymerases during kDNA replication.</p>
},
	keywords = {Cell Cycle, DNA Replication, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Gene Knockdown Techniques, Gene Silencing, Mitochondria, Protozoan Proteins, S Phase, Trypanosoma brucei brucei},
	issn = {1939-4586},
	doi = {10.1091/mbc.E18-02-0127},
	author = {Concepci{\'o}n-Acevedo, Jeniffer and Miller, Jonathan C and Boucher, Michael J and Klingbeil, Michele M}
}
@article {380,
	title = {Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei.},
	journal = {Eukaryot Cell},
	volume = {10},
	year = {2011},
	month = {2011 Jun},
	pages = {734-43},
	abstract = {Trypanosoma brucei, the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite{\textquoteright}s single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei. The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.},
	keywords = {Cell Survival, Cells, Cultured, DNA Replication, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Humans, Membrane Potential, Mitochondrial, Mitochondria, Parasitemia, RNA Interference, Trypanosoma brucei brucei, Trypanosomiasis, African},
	issn = {1535-9786},
	doi = {10.1128/EC.05008-11},
	author = {Bruhn, David F and Sammartino, Mark P and Klingbeil, Michele M}
}
@article {384,
	title = {Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes.},
	journal = {Eukaryot Cell},
	volume = {7},
	year = {2008},
	month = {2008 Dec},
	pages = {2141-6},
	abstract = {Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. 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.},
	keywords = {Animals, DNA Replication, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Mitochondria, Mitochondrial Proteins, Protozoan Proteins, RNA Interference, RNA, Double-Stranded, Trypanosoma brucei brucei},
	issn = {1535-9786},
	doi = {10.1128/EC.00199-08},
	author = {Chandler, Julian and Vandoros, Anthula V and Mozeleski, Brian and Klingbeil, Michele M}
}
@article {386,
	title = {Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes.},
	journal = {J Biol Chem},
	volume = {278},
	year = {2003},
	month = {2003 Dec 5},
	pages = {49095-101},
	abstract = {In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70\% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30\% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.},
	keywords = {Animals, Base Sequence, Cell Line, DNA Polymerase beta, DNA Primers, Isoenzymes, Mitochondria, Recombinant Proteins, Trypanosoma brucei brucei},
	issn = {0021-9258},
	doi = {10.1074/jbc.M308565200},
	author = {Saxowsky, Tina T and Choudhary, Gunjan and Klingbeil, Michele M and Englund, Paul T}
}
@article {388,
	title = {Multiple mitochondrial DNA polymerases in Trypanosoma brucei.},
	journal = {Mol Cell},
	volume = {10},
	year = {2002},
	month = {2002 Jul},
	pages = {175-86},
	abstract = {Kinetoplast DNA (kDNA), the unusual mitochondrial DNA of Trypanosoma brucei, is a network containing thousands of catenated circles. Database searching for a kDNA replicative polymerase (pol) revealed no mitochondrial pol gamma homolog. Instead, we identified four proteins (TbPOLIA, IB, IC, and ID) related to bacterial pol I. Remarkably, all four localized to the mitochondrion. TbPOLIB and TbPOLIC localized beside the kDNA where replication occurs, and their knockdown by RNA interference caused kDNA network shrinkage. Furthermore, silencing of TbPOLIC caused loss of both minicircles and maxicircles and accumulation of minicircle replication intermediates, consistent with a role in replication. While typical mitochondria contain one DNA polymerase, pol gamma, trypanosome mitochondria contain five such enzymes, including the previously characterized pol beta.},
	keywords = {Amino Acid Sequence, Animals, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Mitochondria, Molecular Sequence Data, Phylogeny, Protein Structure, Tertiary, Protein Transport, RNA, Double-Stranded, RNA, Messenger, Sequence Homology, Amino Acid, Trypanosoma brucei brucei},
	issn = {1097-2765},
	author = {Klingbeil, Michele M and Motyka, Shawn A and Englund, Paul T}
}
