Mitochondrial DNA Ligases of
            <i>Trypanosoma brucei</i>

Downey, Nick; Hines, Jane C.; Sinha, Krishna M.; Ray, Dan S.

doi:10.1128/ec.4.4.765-774.2005

Cited by 67 publications

(89 citation statements)

References 36 publications

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“…Others have suggested that the antipodal sites are organized into subdomains populated by different enzyme activities (9,15). Multiple lines of evidence support this view.…”

Section: Discussionsupporting

confidence: 53%

“…Multiple lines of evidence support this view. First, Okazaki fragment-processing enzymes Pol ␤ and SSE1 appear to colocalize (12), while TopoII mt and Ligk ␤ do not precisely colocalize (9). Second, ethanolic-phosphotungstic acid staining showed regions that differ in staining intensity, indicating the variability of protein concentration in different regions of the antipodal sites (15).…”

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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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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“…Others have suggested that the antipodal sites are organized into subdomains populated by different enzyme activities (9,15). Multiple lines of evidence support this view.…”

Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

2012

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“…2D and 5B). In the case of several other kDNA replication proteins silenced by using RNAi (1,(25)(26)(27), it has been found that, although kDNA has not been properly replicated, it has nevertheless gone through a segregation process, which resulted in an uneven segregated networks, creating one cell with a minimal kDNA network and another with a very small nonfunctional kDNA (23). Such a process led to shrinkage of the network and eventually to kDNA loss.…”

Section: Discussionmentioning

confidence: 99%

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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“…Within these sites, subsequent steps of replication occur, catalyzed by enzymes specifically localized therein. These reactions are thought to include primer removal by a structure-specific endonuclease-I (SSE1) (15-17) and repair of some (but not all) of the minicircle gaps by a DNA polymerase ␤ (18, 19) and a DNA ligase (20,21). Finally, the progeny minicircles, still containing at least one nick or gap, are attached to the edge of the kDNA network by an antipodal topoisomerase II (22, 23).…”

mentioning

confidence: 99%

Effects of RNA Interference of Trypanosoma brucei Structure-specific Endonuclease-I on Kinetoplast DNA Replication

Liu

Motyka

Englund

2005

Journal of Biological Chemistry

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Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5 end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell.Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and similar diseases in livestock. Trypanosomes, being one of the earliest branching eukaryotes, have unusual biological properties. Their mitochondrial DNA, known as kinetoplast DNA (kDNA), 2 is one of their most amazing features (see Refs. 1-3 for reviews on kDNA). kDNA is a giant network consisting of topologically interlocked DNA rings. Within the cell, the network is condensed into a disk-shaped structure located within a distended portion of the mitochondrial matrix adjacent to the flagellar basal body. There is a trans-membrane filament system linking the kDNA network to the basal body that facilitates segregation of daughter networks following replication (4, 5).The T. brucei kDNA network contains several thousand minicircles that are heterogeneous in sequence (each 1 kb) and a few dozen maxicircles (each 23 kb). Maxicircles, similar to mitochondrial DNAs of higher organisms, encode rRNAs and subunits of respiratory complexes. Minicircles encode small guide RNAs that control the editing of maxicircle transcripts. Editing is a remarkable process involving the addition or deletion of uridylate residues at specific sites in the transcript to form functional mRNA (reviewed in Refs. 6 and 7).kDNA replication has been studied in T. brucei and also in the related parasite Crithidia fasciculata (reviewed in Refs. 1, 3, and 8). In both species, kDNA synthesis occurs in approximate synchrony with nuclear DNA replication, but division of the kinetoplast occurs prior to that of the nucleus (9, 10). Prior to kDNA replication, during the G 1 phase of the cell cycle, all minicircl...

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Mitochondrial DNA Ligases of Trypanosoma brucei

Cited by 67 publications

References 36 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

Effects of RNA Interference of Trypanosoma brucei Structure-specific Endonuclease-I on Kinetoplast DNA Replication

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