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 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 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 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 Intramitochondrial Location and Dynamics of Crithidia fasciculata Kinetoplast Minicircle Replication Intermediates

2001 ◽  
Vol 153 (4) ◽  
pp. 735-744 ◽  
Author(s):  
Mark E. Drew ◽  
Paul T. Englund
Keyword(s):  
Mitochondrial Dna ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Basal Body ◽  
Mitochondrial Membrane ◽  
Mitochondrial Matrix ◽  
Kinetoplast Dna ◽  
Crithidia Fasciculata ◽  
Replication Intermediates

Kinetoplast DNA, the mitochondrial DNA of Crithidia fasciculata, is organized into a network containing 5,000 topologically interlocked minicircles. This network, situated within the mitochondrial matrix, is condensed into a disk-shaped structure located near the basal body of the flagellum. Fluorescence in situ hybridization revealed that before their replication, minicircles are released vectorially from the network face nearest the flagellum. Replication initiates in the zone between the flagellar face of the disk and the mitochondrial membrane (we term this region the kinetoflagellar zone [KFZ]). The replicating minicircles then move to two antipodal sites that flank the disk-shaped network. In later stages of replication, the number of free minicircles increases, accumulating transiently in the KFZ. The final replication events, including primer removal, repair of many of the gaps, and reattachment of the progeny minicircles to the network periphery, are thought to take place within the antipodal sites.

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.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

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.

scholarly journals Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

1989 ◽  
Vol 9 (11) ◽  
pp. 5163-5168 ◽  
Author(s):  
D M Engman ◽  
L V Kirchhoff ◽  
J E Donelson
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Protozoan Parasite ◽  
Specific Region ◽  
Kinetoplast Dna ◽  
Intracellular Location ◽  
E Coli ◽  
Hsp70 Family ◽  
Mitochondrial Dna Replication ◽  
Initiation Of Dna Replication

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.

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