Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

Bruhn, David F.; Sammartino, Mark P.; Klingbeil, Michele M.

doi:10.1128/ec.05008-11

Cited by 24 publications

(19 citation statements)

References 54 publications

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“…Two distinct regions, the antipodal sites and the KFZ, have emerged as important sites for the localization of kDNA replication proteins and minicircle replication intermediates. Previously, we demonstrated that three mitochondrial DNA polymerases (TbPOLIB, TbPOLIC, and TbPOLID) are essential for parasite survival and kDNA replication in both life cycle stages (4)(5)(6)22). While TbPOLIB and TbPOLIC were detected in the KFZ, TbPOLID did not specifically localize near the kDNA disk (22).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012

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Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. 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.M itochondrial DNA (mtDNA) is packaged into protein-DNA complexes called nucleoids. These structures are dynamic macrocomplexes located in the mitochondrial matrix, and they act as units of mtDNA replication and inheritance with composition that can undergo remodeling in response to metabolic stresses (7,23,47). Using bromodeoxyuridine (BrdU) incorporation, nucleoids have been shown to be the sites of mtDNA replication in yeast and mammalian cells (35,39). However, not all nucleoids replicate concurrently; only a subset undergo replication at any given time. With no strict control related to cell cycle progression, the segregation and inheritance of the nucleoid depends upon a membrane-associated apparatus that interacts with the fusion and fission machinery of the mitochondrial organelle network (2,19,31). Lastly, while the protein composition of nucleoids varies among cell types and in response to metabolic conditions, the core proteins of the nucleoid appear to remain constant and include transcription and replication factors, such as mitochondrial transcription factor A, single-stranded binding protein, Twinkle helicase, and the sole mitochondrial DNA polymerase, Pol ␥ (1, 21).One of the most unusual and structurally complex mtDNA genomes is found in trypanosomatid parasites such as Trypanosoma brucei, the causative agent of African sleeping sickness. This extranuclear genome, called kinetoplast DNA (kDNA), is a network composed of thousands of topologically interlocked minicircles and maxicircles that are condensed into a single diskshaped nucleoid structure. Approximately 25 identical maxicircle copies (23 kb) encode a subset of respiratory c...

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Section: Discussionmentioning

confidence: 99%

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012

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“…brucei gambiense) and animal African trypanosomiasis (AAT) (caused by T. brucei brucei), the presence of a functional kinetoplast is essential (1–3). However, the closely related animal pathogens Trypanosoma equiperdum and Trypanosoma evansi are dyskinetoplastic (DK) (i.e., lacking all or critical parts of their kDNA), with no apparent detrimental effect (4, 5).…”

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Independence from Kinetoplast DNA Maintenance and Expression Is Associated with Multidrug Resistance in Trypanosoma brucei In Vitro

Gould

Schnaufer

2014

Antimicrob Agents Chemother

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It is well known that several antitrypanosomatid drugs accumulate in the parasite's mitochondrion, where they often bind to the organellar DNA, the kinetoplast. To what extent this property relates to the mode of action of these compounds has remained largely unquantified. Here we show that single point mutations that remove the dependence of laboratory strains of the sleeping sickness parasite Trypanosoma brucei on a functional kinetoplast result in significant resistance to the diamidine and phenanthridine drug classes.

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“…Although there are two classes of DNA molecules, there are six different mitochondrial DNA polymerases, the specific functions of which have yet to be completely elucidated (16)(17)(18). Replication of the kDNA network has been studied in detail, especially in the Englund laboratory (19).…”

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Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

et al. 2013

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bThe mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

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Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

Cited by 24 publications

References 54 publications

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Independence from Kinetoplast DNA Maintenance and Expression Is Associated with Multidrug Resistance in Trypanosoma brucei In Vitro

Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

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