scholarly journals Mitochondrial DNA Ligases of Trypanosoma brucei

2005 ◽  
Vol 4 (4) ◽  
pp. 765-774 ◽  
Author(s):  
Nick Downey ◽  
Jane C. Hines ◽  
Krishna M. Sinha ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Topoisomerase Ii ◽  
Leishmania Major ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Dna Ligase ◽  
Kinetoplast Dna ◽  
Rapid Loss ◽  
Dna Ligases

ABSTRACT The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG kα and LIG kβ) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG kα and LIG kβ are mitochondrial DNA ligases. Epitope-tagged LIG kα localizes throughout the kDNA, whereas LIG kβ shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG kα mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.

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 Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

1995 ◽  
Vol 15 (12) ◽  
pp. 6794-6803 ◽  
Author(s):  
L R Carpenter ◽  
P T Englund
Keyword(s):  
Electron Microscopy ◽  
Trypanosoma Brucei ◽  
Topoisomerase Ii ◽  
Repetitive Sequences ◽  
Variable Region ◽  
Kinetoplast Dna ◽  
Crithidia Fasciculata ◽  
Interstrand Cross Links ◽  
Replication Intermediates

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.

scholarly journals Identification of New Kinetoplast DNA Replication Proteins in Trypanosomatids Based on Predicted S-Phase Expression and Mitochondrial Targeting

2007 ◽  
Vol 6 (12) ◽  
pp. 2303-2310 ◽  
Author(s):  
Yue Li ◽  
Yu Sun ◽  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Leishmania Major ◽  
S Phase ◽  
Regulatory Sequences ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Mitochondrial Targeting ◽  
Novel Proteins ◽  
Trypanosomatid Parasites

ABSTRACT Trypanosomatid parasites contain an unusual form of mitochondrial DNA (kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The Leishmania major protein P105 (LmP105) and Trypanosoma brucei protein P93 (TbP93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the LmP105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of TbP93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of TbP93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.

scholarly journals Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

2008 ◽  
Vol 82 (12) ◽  
pp. 5922-5932 ◽  
Author(s):  
Y.-C. James Lin ◽  
Jianhong Li ◽  
Chad R. Irwin ◽  
Heather Jenkins ◽  
Luke DeLange ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Topoisomerase Ii ◽  
Nuclear Dna ◽  
Wild Type Virus ◽  
Dna Ligase ◽  
Topoisomerase Iiα ◽  
Bait Protein ◽  
Y Substitution ◽  
In Cells

ABSTRACT Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIβ. One can prevent the interaction by introducing a C11-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIα/β in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIα and IIβ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIα/β in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.

scholarly journals Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

2011 ◽  
Vol 10 (6) ◽  
pp. 734-743 ◽  
Author(s):  
David F. Bruhn ◽  
Mark P. Sammartino ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dna Polymerases ◽  
Bloodstream Form ◽  
Complex Life Cycle ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Parasite Viability

ABSTRACT Trypanosoma brucei , the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei . The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.

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.

scholarly journals Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei

2014 ◽  
Vol 42 (15) ◽  
pp. 9717-9729 ◽  
Author(s):  
David Reynolds ◽  
Laura Cliffe ◽  
Konrad U. Förstner ◽  
Chung-Chau Hon ◽  
T. Nicolai Siegel ◽  
...  
Keyword(s):  
Cell Death ◽  
Trypanosoma Brucei ◽  
Leishmania Major ◽  
Nuclear Dna ◽  
Epigenetic Modification ◽  
Transcription Termination ◽  
Gene Clusters ◽  
Regulation Of Transcription ◽  
Genome Wide ◽  
Rnap Ii

Abstract Base J, β-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase (RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.

scholarly journals Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

2013 ◽  
Vol 12 (7) ◽  
pp. 970-978 ◽  
Author(s):  
Kirsten Beck ◽  
Nathalie Acestor ◽  
Anjelique Schulfer ◽  
Atashi Anupama ◽  
Jason Carnes ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Rna Interference ◽  
Fluorescence Microscopy ◽  
Trypanosoma Brucei ◽  
Cell Population ◽  
Growth Defect ◽  
Kinetoplast Dna ◽  
Multiprotein Complexes ◽  
Proteomic Approach ◽  
Novel Protein

ABSTRACT The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

scholarly journals Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria

2021 ◽  
Author(s):  
Benoit Roch ◽  
Ekaterina Pisareva ◽  
Cynthia Sanchez ◽  
Brice Pastor ◽  
Rita Tanos ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Extracellular Vesicles ◽  
High Speed ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Structural Features ◽  
Differential Centrifugation ◽  
Analytical Strategy ◽  
Low Pass ◽  
Physical Examinations

Circulating mitochondrial DNA (cir-mtDNA) could have a potential comparable to circulating nuclear DNA (cir-nDNA), with numerous applications. However, research and development in this area have fallen behind, particularly considering its origin and structural features. To tackle this, we initially combined Q-PCR and low-pass whole genome sequencing in the same analytical strategy previously and successfully used for cir-nDNA. This revealed unexplained structural patterns and led us to correlate these data with observations made during physical examinations such as filtration, and differential centrifugation in various plasma preparations. Both the integrity index and number of reads revealed a very minor proportion of low size-ranged fragments (<1000 bp) in plasma obtained with a standard preparation (0.06%). Filtration and high speed second step centrifugation revealed that 98.7 and 99.4% corresponded to extracellular mitochondria either free or in large extracellular vesicles. When avoiding platelet activation during plasma preparation, the proportion of both types of entities was still preponderant (76-80%), but the amount of detected mitochondrial DNA decreased 67-fold. In correlation with our previous study on the presence of circulating cell-free mitochondria in blood, our differential centrifugation procedure suggested that cir-mtDNA is also associated with approximately 18% small extracellular vesicles, 1.7% exosomes and 4% protein complexes.

The replication mechanism of kinetoplast DNA networks in several trypanosomatid species

1998 ◽  
Vol 111 (6) ◽  
pp. 675-679
Author(s):  
D.L. Guilbride ◽  
P.T. Englund
Keyword(s):  
Dna Replication ◽  
Fluorescence Microscopy ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Topoisomerase Ii ◽  
Leishmania Donovani ◽  
Kinetoplast Dna ◽  
Crithidia Fasciculata ◽  
Replication Mechanism ◽  
Novel Method

Kinetoplast DNA, a giant network of interlocked DNA circles, replicates by an unusual mechanism. Minicircles are released individually from the network by a topoisomerase II, and then, after replication, their progeny are reattached at antipodal positions on the network periphery. Studies to date have revealed two distinct variations on this model. In Crithidia fasciculata the newly replicated minicircles quickly become uniformly distributed around the network periphery, whereas in Trypanosoma brucei the minicircles accumulate near their two points of attachment. The kinetoplast DNA replication mechanism used by other related trypanosomatid species was until now unknown. Here we used a novel method, involving fluorescence microscopy of isolated networks, to investigate kinetoplast DNA replication in Leishmania tarentolae, Leishmania donovani, Trypanosoma cruzi and Phytomonas serpens. We found that all of these species have a replication mechanism resembling that of C. fasciculata and that the polar replication mechanism observed in T. brucei is so far unique to this species.

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