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.

Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation

1994 ◽  
Vol 14 (6) ◽  
pp. 3660-3667
Author(s):  
T A Shapiro
Keyword(s):  
Replication Fork ◽  
Topoisomerase Ii ◽  
Potent Inhibitor ◽  
Late Replication ◽  
Kinetoplast Dna ◽  
Two Dimensional Gel Electrophoresis ◽  
Trypanosoma Equiperdum ◽  
Replication Intermediates ◽  
Novel Structures

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.

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 The structure of replicating kinetoplast DNA networks.

1993 ◽  
Vol 123 (5) ◽  
pp. 1069-1079 ◽  
Author(s):  
D Pérez-Morga ◽  
P T Englund
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Structural Changes ◽  
Kinetoplast Dna ◽  
Nick Translation ◽  
Crithidia Fasciculata ◽  
Intercalating Dye

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.

scholarly journals Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation.

1994 ◽  
Vol 14 (6) ◽  
pp. 3660-3667 ◽  
Author(s):  
T A Shapiro
Keyword(s):  
Replication Fork ◽  
Topoisomerase Ii ◽  
Potent Inhibitor ◽  
Late Replication ◽  
Kinetoplast Dna ◽  
Two Dimensional Gel Electrophoresis ◽  
Trypanosoma Equiperdum ◽  
Replication Intermediates ◽  
Novel Structures

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.

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.

Localization of kinetoplast DNA maxicircle transcripts in bloodstream and procyclic form Trypanosoma brucei

1982 ◽  
Vol 2 (7) ◽  
pp. 845-852
Author(s):  
K D Stuart ◽  
S B Gelvin
Keyword(s):  
Restriction Endonuclease ◽  
Trypanosoma Brucei ◽  
Variable Region ◽  
Cleavage Sites ◽  
Kinetoplast Dna ◽  
Coding Sequences ◽  
Procyclic Form ◽  
Restriction Endonuclease Cleavage ◽  
Mitochondrial Respiratory

Over 80% of the maxicircle and numerous minicircles of Trypanosoma brucei kinetoplast DNA have been cloned. The uncloned maxicircle segment contains few restriction endonuclease cleavage sites, varies in size among strains, and may be unstable in conventional cloning systems. cDNA prepared to bloodstream or procyclic trypomastigote RNA hybridized to all but one maxicircle segment, but did not hybridize to minicircles. Fourteen maxicircle transcripts were detected in RNA from both bloodstream and procyclic trypomastigotes. The coding sequences for these transcripts were localized and account for most of the maxicircle. One region of the maxicircle, which borders the variable region, was not found to be transcribed. We conclude that the maxicircle is largely but not completely transcribed in both bloodstream and procyclic trypomastigotes, whereas minicircle transcription is minimal or absent in these stages. Qualitative transcriptional differences which could account for mitochondrial respiratory differences between the bloodstream and procyclic trypomastigotes were not observed.

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 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

Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles

1989 ◽  
Vol 9 (1) ◽  
pp. 169-176
Author(s):  
C Sheline ◽  
T Melendy ◽  
D S Ray
Keyword(s):  
Topoisomerase Ii ◽  
Short Pulse ◽  
Kinetoplast Dna ◽  
Crithidia Fasciculata ◽  
Open Circular ◽  
Leading Strand ◽  
Polymerase I ◽  
Open Circular Form ◽  
Lagging Strand ◽  
Migration Component

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.

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