scholarly journals Role of p38 in Replication of Trypanosoma brucei Kinetoplast DNA

2006 ◽  
Vol 26 (14) ◽  
pp. 5382-5393 ◽  
Author(s):  
Beiyu Liu ◽  
Henrik Molina ◽  
Dario Kalume ◽  
Akhilesh Pandey ◽  
Jack D. Griffith ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dna Replication ◽  
Mitochondrial Genome ◽  
Dna Synthesis ◽  
Trypanosoma Brucei ◽  
Replication Origin ◽  
Mitochondrial Protein ◽  
Kinetoplast Dna ◽  
Z Dna

ABSTRACT Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as θ-structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.

scholarly journals The Double-Edged Sword in Pathogenic Trypanosomatids: The Pivotal Role of Mitochondria in Oxidative Stress and Bioenergetics

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Rubem Figueiredo Sadok Menna-Barreto ◽  
Solange Lisboa de Castro
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Signalling ◽  
Oxygen Species ◽  
Kinetoplast Dna ◽  
Excellent Candidate ◽  
Causative Agents ◽  
Parasite Biology ◽  
Oxidative Regulation ◽  
Single Mitochondrion

The pathogenic trypanosomatidsTrypanosoma brucei,Trypanosoma cruzi, andLeishmaniaspp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids’ life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes.

Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei

1990 ◽  
Vol 95 (1) ◽  
pp. 49-57 ◽  
Author(s):  
R. Woodward ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Unit Cell ◽  
Kinetoplast Dna ◽  
Interphase Cell ◽  
Body Formation ◽  
Timing Of Initiation ◽  
Single Mitochondrion

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

The programme of DNA replication: beyond genome duplication

2013 ◽  
Vol 41 (6) ◽  
pp. 1720-1725 ◽  
Author(s):  
Blanca Gómez-Escoda ◽  
Pei-Yun Jenny Wu
Keyword(s):  
Dna Replication ◽  
Cell Growth ◽  
Dna Synthesis ◽  
Genetic Information ◽  
Replication Origin ◽  
Genome Duplication ◽  
Replication Initiation ◽  
Origin Activation ◽  
Key Steps ◽  
Cell Growth And Proliferation

The accurate duplication and transmission of genetic information is critical for cell growth and proliferation, and this is ensured in part by the multi-layered regulation of DNA synthesis. One of the key steps in this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, origin usage changes during development and in different pathologies, suggesting an integral interplay between the establishment of replication initiation along the chromosomes and cellular function. The present review discusses how the spatiotemporal organization of replication origin activation may play crucial roles in the control of biological events.

scholarly journals Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea

2009 ◽  
Vol 8 (10) ◽  
pp. 1592-1603 ◽  
Author(s):  
Patrícia Diogo de Melo Godoy ◽  
Luis Antonio Nogueira-Junior ◽  
Lisvane S. Paes ◽  
Alberto Cornejo ◽  
Rafael Miyazawa Martins ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Replication Origin ◽  
Origin Recognition Complex ◽  
Gene Encoding ◽  
Unicellular Eukaryotes ◽  
Entire Cell ◽  
Multicellular Organisms

ABSTRACT In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

scholarly journals A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

2017 ◽  
Author(s):  
Anneliese Hoffmann ◽  
Sandro Käser ◽  
Martin Jakob ◽  
Simona Amodeo ◽  
Camille Peitsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
De Novo ◽  
Molecular Model ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Kinetoplast Dna ◽  
Exclusion Zone ◽  
Super Resolution Microscopy

AbstractIn almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation.Significance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.Abbreviation(EZF)Exclusion zone filaments(ULF)Unilateral filament(TAC)tripartite attachment complex(OM)outer mitochondrial(IM)inner mitochondrial(BSF)bloodstream form(PCF)procyclic form(kDNA)kinetoplast DNA(gRNA)guide RNA(SBFSEM)Serial block face-scanning electron microscopy(Tet)tetracyclin(STED)Stimulated Emission Depletion

scholarly journals Effect of Hydroxyurea on Procyclic Trypanosoma brucei: an Unconventional Mechanism for Achieving Synchronous Growth

2007 ◽  
Vol 7 (2) ◽  
pp. 425-428 ◽  
Author(s):  
Arnab Roy Chowdhury ◽  
Zhixing Zhao ◽  
Paul T. Englund
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
S Phase ◽  
Kinetoplast Dna ◽  
Synchronous Growth

ABSTRACT Procyclic Trypanosoma brucei cells were synchronized with 0.2 mM hydroxyurea. The cells did not arrest at the G1/S boundary but proceeded through one round of replication and arrested near the end of S phase. The mitochondrial genome (kinetoplast DNA network) replicated, forming two progeny networks, but the repair of minicircle gaps was inhibited.

scholarly journals Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata.

1994 ◽  
Vol 126 (3) ◽  
pp. 631-639 ◽  
Author(s):  
M L Ferguson ◽  
A F Torri ◽  
D Pérez-Morga ◽  
D C Ward ◽  
P T Englund
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Protein Complexes ◽  
Kinetoplast Dna ◽  
Relative Movement ◽  
Crithidia Fasciculata ◽  
Trypanosomatid Parasites

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.

scholarly journals A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei

2010 ◽  
Vol 30 (6) ◽  
pp. 1319-1328 ◽  
Author(s):  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Cell Growth ◽  
Trypanosoma Brucei ◽  
Dna Polymerase ◽  
Rnase H ◽  
Kinetoplast Dna ◽  
African Trypanosomes ◽  
Dna Primase

ABSTRACT Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3′-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

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