Role of p38 in Replication of <i>Trypanosoma brucei</i> Kinetoplast DNA

Liu, Beiyu; Molina, Henrik; Kalume, Dário Eluan; Pandey, Akhilesh; Griffith, Jack D.; Englund, Paul T.

doi:10.1128/mcb.00369-06

Cited by 53 publications

(94 citation statements)

References 47 publications

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“…Another possibility is that this process is not directed by the UMSBP-bound origin but by an alternative initiation site in the minicircle molecule. It is also possible that other origin-binding proteins, such as the T. brucei p38, which binds the minicircle replication origin (24), could partially substitute for the loss of UMSBP function.…”

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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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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“…Cells were fixed in paraformaldehyde, permeabilized in methanol, and labeled with terminal deoxynucleotidyl transferase (TdT) as previously described (20,26). Briefly, cells were rehydrated in PBS and incubated for 20 min at room temperature in 25 l of 1ϫ TdT reaction buffer (Roche Applied Science) containing 2 mM CoCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…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). The free minicircles initiate unidirectional theta structure replication primarily through interactions with proteins such as universal minicircle sequence binding protein (UMSBP) and p38 (26,33). TbPOLIB and TbPOLIC also localize to this region.…”

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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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“…However, a recent study of REAP-1 knock-out cells implies a role in RNA stability rather than editing (Hans et al 2007). Although RBP38 was first identified as a protein that binds gRNA-mRNA duplexes (Sbicego et al 2003), its function seems to be in the initiation of kinetoplast DNA replication (Liu et al 2006).…”

Section: Introductionmentioning

confidence: 99%

TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

Hashimi¹,

Zı́ková²,

Panigrahi³

et al. 2008

RNA

144

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The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1).

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Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

Cited by 53 publications

References 47 publications

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

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

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

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