@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 {1274,
	title = {Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching.},
	journal = {Mol Microbiol},
	volume = {87},
	year = {2013},
	month = {2013 Jan},
	pages = {196-210},
	abstract = {<p>Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host\&$\#$39;s immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T.\&thinsp;brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T.\&thinsp;brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.</p>
},
	keywords = {Antigenic Variation, DNA Replication, DNA, Protozoan, Gene Silencing, Genes, Protozoan, Membrane Glycoproteins, Origin Recognition Complex, Promoter Regions, Genetic, Trypanosoma brucei brucei, Variant Surface Glycoproteins, Trypanosoma},
	issn = {1365-2958},
	doi = {10.1111/mmi.12093},
	author = {Benmerzouga, Imaan and Concepci{\'o}n-Acevedo, Jeniffer and Kim, Hee-Sook and Vandoros, Anthula V and Cross, George A M and Klingbeil, Michele M and Li, Bibo}
}
@article {381,
	title = {Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei.},
	journal = {Mol Biochem Parasitol},
	volume = {175},
	year = {2011},
	month = {2011 Jan},
	pages = {68-75},
	abstract = {The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.},
	keywords = {Cell Nucleus, Dyneins, Flagella, Locomotion, Microscopy, Electron, Transmission, Organelles, Protozoan Proteins, RNA Interference, Trypanosoma brucei brucei},
	issn = {1872-9428},
	doi = {10.1016/j.molbiopara.2010.09.005},
	author = {Springer, Amy L and Bruhn, David F and Kinzel, Kathryn W and Rosenthal, No{\"e}l F and Zukas, Randi 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 {382,
	title = {Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes.},
	journal = {Mol Microbiol},
	volume = {75},
	year = {2010},
	month = {2010 Mar},
	pages = {1414-25},
	abstract = {The unique mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles called kinetoplast DNA (kDNA). The network is essential for survival, and requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication. At least seven DNA polymerases (pols) are involved in kDNA transactions, including three essential proteins related to bacterial DNA pol I (POLIB, POLIC and POLID). How Trypanosoma brucei utilizes multiple DNA pols to complete the topologically complex task of kDNA replication is unknown. To fill this gap in knowledge we investigated the cellular role of POLIB using RNA interference (RNAi). POLIB silencing resulted in growth inhibition and progressive loss of kDNA networks. Additionally, unreplicated covalently closed precursors become the most abundant minicircle replication intermediate as minicircle copy number declines. Leading and lagging strand minicircle progeny similarly declined during POLIB silencing, indicating POLIB had no apparent strand preference. Interestingly, POLIB RNAi led to the accumulation of a novel population of free minicircles that is composed mainly of covalently closed minicircle dimers. Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery.},
	keywords = {DNA Polymerase beta, DNA Replication, DNA, Kinetoplast, Gene Silencing, Protozoan Proteins, Trypanosoma brucei brucei},
	issn = {1365-2958},
	doi = {10.1111/j.1365-2958.2010.07061.x},
	author = {Bruhn, David F and Mozeleski, Brian and Falkin, Laurie and Klingbeil, Michele M}
}
@article {383,
	title = {Unraveling the secrets of regulating mitochondrial DNA replication.},
	journal = {Mol Cell},
	volume = {35},
	year = {2009},
	month = {2009 Aug 28},
	pages = {398-400},
	abstract = {In this issue, Liu et al. (2009) report that maxicircle DNA copy number in trypanosomes is regulated by proteolysis of a helicase; the complex kinetoplast DNA system yields a clear view of how mitochondrial DNA replication can be regulated.},
	keywords = {Animals, DNA Helicases, DNA Replication, DNA, Kinetoplast, DNA, Mitochondrial, DNA, Protozoan, Gene Expression Regulation, Mutation, Peptide Hydrolases, Protozoan Proteins, Time Factors, Trypanosoma brucei brucei},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2009.08.007},
	author = {Klingbeil, Michele M and Shapiro, Theresa A}
}
@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 {385,
	title = {Closing the gaps in kinetoplast DNA network replication.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {101},
	year = {2004},
	month = {2004 Mar 30},
	pages = {4333-4},
	keywords = {Animals, Crithidia fasciculata, DNA Ligases, DNA Replication, DNA, Kinetoplast, DNA, Mitochondrial, Microscopy, Electron, Trypanosoma brucei brucei},
	issn = {0027-8424},
	doi = {10.1073/pnas.0401400101},
	author = {Klingbeil, Michele M and Englund, Paul T}
}
@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}
}
