Multiple Mitochondrial DNA Polymerases in Trypanosoma brucei

Klingbeil, Michele M.; Motyka, Shawn A.; Englund, Paul T.

doi:10.1016/s1097-2765(02)00571-3

Cited by 124 publications

(180 citation statements)

References 41 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: 88%

“…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). Given its essential role in kDNA replication, the localization of TbPOLID to the mitochondrial matrix suggested major differences in the mechanism of how this protein engaged in kDNA replication.…”

Section: Discussionmentioning

confidence: 92%

“…Previously, we demonstrated that TbPOLID is one of three mitochondrial DNA polymerases that are essential for parasite survival and the maintenance of the kDNA network (6). However, the initial localization of TbPOLID using a peptide antibody showed that it was distributed throughout the mitochondrial matrix (22), suggesting that POLID would need to redistribute to the kDNA disk to perform its essential role in kDNA replication. To investigate the localization of TbPOLID in detail, we generated an exclusive expresser cell line, TbID-PTP, in which one TbPOLID allele was deleted and the remaining allele was fused to the PTP sequence by the targeted integration of the construct pPOLID-PTP-NEO (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…RNA editing depends upon guide RNAs (gRNAs) encoded on the heterogeneous population of 5,000 minicircles (1 kb) (49). Therefore, the coordinated replication of both minicircles and maxicircles is essential for mitochondrial physiology and parasite survival.Key features of the kDNA replication mechanism include replication once per cell cycle in near synchrony with nuclear S phase, a topoisomerase II-mediated release and reattachment of minicircles, and a multiplicity of DNA polymerases (six), helicases (six), and primases (two) for kDNA transactions (17,18,22,27,45). Briefly, covalently closed minicircles are released from the network into the kinetoflagellar zone (KFZ), a specialized region between the kDNA disk and the mitochondrial membrane nearest the basal body (bb) (10).…”

mentioning

confidence: 99%

“…It has been proposed that the composition of proteins at the antipodal sites is dynamic, and the localization of some of these proteins to the antipodal sites seems to be periodic (20,46). Initial localization studies of the three essential mitochondrial DNA polymerases indicated that TbPOLIB and TbPOLIC localized to the KFZ, while TbPOLID was distributed throughout the mitochondrial matrix (22). The matrix localization of TbPOLID suggests that this protein redistributes close to the kDNA network or is needed in very low abundance to perform its role in kDNA replication.…”

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confidence: 99%

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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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