Trypanosomes Have Six Mitochondrial DNA Helicases with One Controlling Kinetoplast Maxicircle Replication

Liu, Beiyu; Wang, Jianyang; Yaffe, Nurit; Lindsay, Megan E.; Zhao, Zhixing; Zick, Aviad; Shlomai, Joseph; Englund, Paul T.

doi:10.1016/j.molcel.2009.07.004

Cited by 64 publications

(114 citation statements)

References 44 publications

(68 reference statements)

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“…The only mitochondrial protease that has been shown to regulate kDNA replication is TbHslVU (25). This bacterial-like protease regulates maxicircle replication through the degradation of the helicase TbPIF2 (27). To determine if proteolytic degradation plays a role in the regulation of POLID-PTP localization patterns, we inhibited protein synthesis using 100 g/ml of CHX during a time course of 6 h. For this experiment we generated a cell line coexpressing POLID-PTP and PIF2-Myc, and we monitored protein levels by immunoblotting.…”

Section: Resultsmentioning

confidence: 99%

“…These data suggested that POLID also has an important role in maxicircle replication (6). So far, only two proteins have definitive roles in maxicircle replication, the primase PRI1 and the helicase PIF2 (17,27). Significant questions about maxicircle replication remain unanswered, including what is the maxicircle replicase.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

“…In contrast to minicircles, maxicircles remain catenated to the network during replication, accumulate at the center of a growing disk, and are the last molecules to segregate (14). Currently, only two proteins have been shown to be essential for maxicircle replication, a DNA helicase (TbPIF2) and a primase (PRI1) (17,27). Thus far, the main maxicircle replicase has not been described.…”

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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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012

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“…A plethora of proteins involved in kDNA replication have been studied in T. brucei, including six helicases (25)(26)(27)40), two DNA ligases (10), two primases (19,20), a topoisomerase IA (40), a topoisomerase II (50), and five DNA polymerases (Pols) (4,7,21,35). The involvement of multiple DNA polymerases in kDNA replication distinguishes this process from replication of other mitochondrial genomes, which depend solely upon DNA polymerase ␥, a family A DNA polymerase.…”

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confidence: 99%