Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Saxowsky, Tina T.; Choudhary, Gunjan; Klingbeil, Michele M.; Englund, Paul T.

doi:10.1074/jbc.m308565200

Cited by 80 publications

(94 citation statements)

References 29 publications

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“…A great majority of newly identified essential kDNA replication proteins with roles in origin binding, priming, and processive replication also localize to the antipodal sites (p38, p93, PRI1, PRI2, PIF1, and PIF5), suggesting that the role of the antipodal sites is not limited to Okazaki fragment processing. Interestingly, several antipodal site proteins appear to transiently associate with this region, demonstrating that the protein composition is dynamic (20,43,46). Studies from the related trypanosome Crithidia fasiculata established that the antipodal localization of Pol ␤ and SSE1 correlated with kDNA synthesis and not other cell cycle stages (12,20).…”

Section: Discussionmentioning

confidence: 99%

“…Minicircle progeny (still containing at least one gap) are subsequently reattached at two electron-dense areas positioned 180°apart at the network periphery called the antipodal sites. Several proteins associated with Okazaki fragment processing and reattachment to the network localize to the antipodal sites, including SSE1, DNA Pol ␤, DNA ligase k␤ (Ligk ␤), and topoisomerase II (Topo II mt ) (9,12,20,43,50). As a result, there is spatial and temporal separation of replication events with early initiation occurring in the KFZ, followed by Okazaki fragment processing and reattachment at the antipodal sites.…”

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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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Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012

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“…To study the status of gap-filling of minicircles in the giant kDNA networks, we have used the in situ incorporation of fluorescently labeled dUTP into kDNA networks by terminal deoxynucleotidyl transferase (TdT) (16). Under normal growth conditions, the final gap filling and sealing of replicated minicircles occurs after their reattachment to the kDNA network, possibly through the action of DNA polymerase ␤-PAK and ligase k␣ (17)(18)(19). Indeed, in uninduced cells, most of the kDNA networks were of an apparently normal size and were negative for the dUTP fluorescence signal, whereas larger kDNA networks display a fluorescence signal, indicating that they were replicating networks containing gapped minicircles (Fig.…”

Section: Unsegregated Network In Tbumsbps-silenced Cells Contain Gappedmentioning

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Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

Milman

Motyka

Englund

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.kDNA replication initiation ͉ kDNA segregation ͉ kinetoplast DNA ͉ trypanosomes cell cycle control ͉ universal minicircle sequence-binding protein K inetoplast DNA (kDNA) is a unique extrachromosomal DNA, found in the single mitochondrion of trypanosomatids. It consists, in the different species, of a few dozen maxicircles (20-40 kb each) and a few thousand minicircles (0.5-10 kb each), that are interlocked topologically into a DNA network (1, 2). Minicircles in most trypanosomatid species contain two short sequences that are associated with replication initiation: a dodecamer, designated the universal minicircle sequence (UMS), and a hexamer. These sequences have been mapped to the replication origins of the minicircle's light (L) and heavy (H) strands, respectively. kDNA replication occurs during S phase of the cell cycle, approximately in parallel with nuclear DNA replication (3). Minicircles are released from the network into the kinetoflagellar zone, located in the mitochondrial matrix, between the kDNA network and the flagellar basal body. Each minicircle replicates as an individual replicon, forming gapped and nicked progeny molecules. Minicircle replication intermediates then migrate onto two antipodal sites, flanking the kDNA disk, in which primer-removal, repair of the gaps between Okazaki fragments and reattachment of the progeny minicircles to the network occurs. The final gap-filling and sealing of the topologically linked minicircles take place before the network division (recently reviewed in refs. 1 and 2).We have previously reported the presence in Crithidia fasciculata of a UMS-binding protein (UMSBP). The protein, which contains five tandemly arranged CCHC-type zinc-finger motifs, has been purified from cell extracts, and its encoding gene and genomic locus were cloned and analyzed (4-7). Genes encoding orthologous proteins have been identified in other trypanosomatid species [ref. 8 and supporting information (SI) Table 1]. UMSBP binds specifically the two conserved sequences, located at the minicircle replication origins: the UMS dodecamer and an H-st...

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“…The mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles named kinetoplast DNA. Trypanosoma Pol ␤ proteins may have distinct and nonredundant roles in kDNA replication or maintenance (55,56).…”

Section: Different Classes Of Ber Enzymesmentioning

confidence: 99%

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

Genois

Paquet

Laffitte

et al. 2014

Microbiol Mol Biol Rev

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SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.

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Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Cited by 80 publications

References 29 publications

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

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