Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

Melendy, Thomas; Sheline, Christian T.; Ray, Dan S.

doi:10.1016/0092-8674(88)90252-8

Cited by 144 publications

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“…Originally, the Okazaki fragment-processing proteins, Pol ␤ and SSE1, as well as TopoII mt , were localized to the antipodal sites (12,13,32). 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.…”

Section: Discussionmentioning

confidence: 99%

“…So far, a majority of kDNA replication proteins localize mainly to the KFZ and the antipodal sites. Since the discovery of the first antipodal kDNA replication protein, Topo II mt (32), many more proteins share a pattern of antipodal localization. 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).…”

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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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“…It was found subsequently (13) that minicircles are replicated free of the network and then reattached at the antipodal sites. The finding of a kinetoplast-specific DNA topoisomerase II located at antipodal sites flanking the kDNA disk provided a biochemical basis for the rejoining of newly replicated minicircles at these sites (14). Recent experiments with Trypanosoma brucei using the RNA-interference technique have provided strong support for the involvement of the homologous T. brucei topoisomerase II in rejoining newly replicated minicircles to the kDNA network (15).…”

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“…Surprisingly, the replication proteins identified thus far show unexpected variations in their temporal and spatial localizations. After the initial discovery of the antipodal localization of the kinetoplast type II DNA topoisomerase (14), a ␤-type DNA polymerase (24), and a structure-specific endonuclease (SSE1) with RNase H activity (25) were found to localize at the antipodal sites along with nascent minicircles. SSE1 and polymerase ␤ immunofluorescence are observed at the antipodal sites during kDNA replication, but they disappear shortly after S phase (25,26).…”

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Mitochondrial DNA ligase in Crithidia fasciculata

Sinha

Hines

Downey

2004

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

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Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase ␤, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the ␤ polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).T he mitochondrial DNA of trypanosomatid protozoa (kinetoplast DNA, or kDNA) consists of two forms of topologically interlocked circular DNAs, minicircles, and maxicircles (for recent reviews of kDNA structure and replication, see refs. 1-3). The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each. The maxicircles encode genes for mitochondrial proteins and ribosomal RNAs. Minicircles encode small guide RNAs that serve as templates for RNA editing of maxicircle transcripts (4). In vivo, the kDNA exists as a condensed disk structure 1 m in diameter and Ϸ0.4 m thick. Each minicircle in the kDNA disk is linked to three other minicircles on average and is relaxed, unlike most other circular DNAs in nature, which are negatively supercoiled (5). Electron microscopic observation of sections through the kDNA disk shows fibers aligned parallel to the axis of the disk. This observation together with the thickness of the disk corresponding to approximately one half of the minicircle circumference (6) suggests that individual minicircles may have a topology like that of a rubber band stretched taut from opposite sides of the circle (7). Maxicircles appear to be present throughout the kDNA and exist as a catenated network within the overall network (8). The kDNA isolated from nonreplicating C. fasciculata is a planar elliptical network of interlocked circles with dimensions of Ϸ10 by 15 m.The kDNA disk is always located at the base of the flagellum, with the flagellum perpendicular to the face of the disk. Recent studies (9) have shown that in trypanosomes the kDNA disk is connected physically to the flagellar basal bodies and is segregated by them at cell division. The attachment was found to involve three components that penetrate the inner and outer mitochondrial membranes to link the kDNA physically ...

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“…Here they encounter SSE1 (10), an enzyme with RNase H activity that can remove RNA primers (11), and DNA polymerase ␤ (12), which presumably fills in most of the gaps between Okazaki fragments. Once this processing takes place, the newly replicated minicircles are reattached to the periphery of the kDNA network by a topoisomerase II (13) that also localizes to the antipodal sites (14). Interestingly, one or two gaps are retained on the newly replicated minicircles after they have been reattached to the network (15,16).…”

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Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

Saxowsky¹,

Choudhary²,

Klingbeil³

et al. 2003

Journal of Biological Chemistry

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In higher eukaryotes, DNA polymerase (pol) ␤ resides in the nucleus and participates primarily in DNA repair. The DNA polymerase ␤ 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 ␤-like proteins. One is ϳ70% identical to the C. fasciculata pol ␤ and is likely the homolog of this enzyme. The other, although ϳ30% 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 MgCl 2 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.

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Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

Cited by 144 publications

References 20 publications

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

Mitochondrial DNA ligase in Crithidia fasciculata

Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

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