A gap at a unique location in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum.

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.

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Synthesis and processing of kinetoplast DNA minicircles in Trypanosoma equiperdum

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3212-3217.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3212-3217

Author(s):

K A Ryan ◽

P T Englund

Keyword(s):

Mitochondrial Dna ◽

Gel Electrophoresis ◽

Agarose Gel ◽

Two Dimensional ◽

Agarose Gel Electrophoresis ◽

Kinetoplast Dna ◽

Gap Filling ◽

Synthesis And Processing ◽

Trypanosoma Equiperdum

Kinetoplast DNA, the mitochondrial DNA in trypanosomes, is a giant network containing topologically interlocked minicircles. Replication occurs on free minicircles that have been detached from the network. In this paper, we report studies on the synthesis and processing of the minicircle L and H strands. Analysis of free minicircles from Trypanosoma equiperdum by two-dimensional agarose gel electrophoresis indicated that elongating L strands are present on theta structures. Hybridization studies indicated that L-strand elongation is continuous and unidirectional, starting near nucleotide 805 and proceeding around the entire minicircle. The theta structures segregate into monomeric progeny minicircles, and those with a newly synthesized L strand have a 8-nucleotide gap between nucleotides 805 and 814 (J. M. Ntambi, T. A. Shapiro, K. A. Ryan, and P. T. Englund, J. Biol. Chem. 261:11890-11895, 1986). These molecules are reattached to the network, where repair of the gap takes place. Of the molecules labeled during a 10-min pulse with [3H]thymidine, gap filling occurred on half within about 15 min and on virtually all by 60 min; however, there was no detectable covalent closure of the newly synthesized L strand by 60 min.

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Synthesis and processing of kinetoplast DNA minicircles in Trypanosoma equiperdum.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3212 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3212-3217 ◽

Cited By ~ 25

Author(s):

K A Ryan ◽

P T Englund

Keyword(s):

Mitochondrial Dna ◽

Gel Electrophoresis ◽

Agarose Gel ◽

Two Dimensional ◽

Agarose Gel Electrophoresis ◽

Kinetoplast Dna ◽

Gap Filling ◽

Synthesis And Processing ◽

Trypanosoma Equiperdum

Kinetoplast DNA, the mitochondrial DNA in trypanosomes, is a giant network containing topologically interlocked minicircles. Replication occurs on free minicircles that have been detached from the network. In this paper, we report studies on the synthesis and processing of the minicircle L and H strands. Analysis of free minicircles from Trypanosoma equiperdum by two-dimensional agarose gel electrophoresis indicated that elongating L strands are present on theta structures. Hybridization studies indicated that L-strand elongation is continuous and unidirectional, starting near nucleotide 805 and proceeding around the entire minicircle. The theta structures segregate into monomeric progeny minicircles, and those with a newly synthesized L strand have a 8-nucleotide gap between nucleotides 805 and 814 (J. M. Ntambi, T. A. Shapiro, K. A. Ryan, and P. T. Englund, J. Biol. Chem. 261:11890-11895, 1986). These molecules are reattached to the network, where repair of the gap takes place. Of the molecules labeled during a 10-min pulse with [3H]thymidine, gap filling occurred on half within about 15 min and on virtually all by 60 min; however, there was no detectable covalent closure of the newly synthesized L strand by 60 min.

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The kinetoplast DNA of Trypanosoma equiperdum

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis ◽

10.1016/0005-2787(80)90150-1 ◽

1980 ◽

Vol 607 (3) ◽

pp. 397-410 ◽

Cited By ~ 44

Author(s):

A.C.C. Frasch ◽

S.L. Hajduk ◽

J.H.J. Hoeijmakers ◽

P. Borst ◽

F. Brunel ◽

...

Keyword(s):

Kinetoplast Dna ◽

Trypanosoma Equiperdum

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Dyskinetoplasty in two species of trypanosomatids

Journal of Cell Science ◽

10.1242/jcs.35.1.185 ◽

1979 ◽

Vol 35 (1) ◽

pp. 185-202

Author(s):

S.L. Hajduk

Keyword(s):

Fluorescence Microscopy ◽

Kinetoplast Dna ◽

Feulgen Staining ◽

Crithidia Fasciculata ◽

Caesium Chloride ◽

Electron And Fluorescence Microscopy ◽

Staining Techniques ◽

Trypanosoma Equiperdum ◽

In Cells

Dyskinetoplastic cells from both Crithidia fasciculata and Trypanosoma equiperdum lack detectable kinetoplast DNA (kDNA) by conventional staining techniques. Two dyskinetoplastic strains of T. equiperdum, either acriflavine-induced or spontaneously occurring, show normal amounts of kDNA (p = 1.692 g/cm3) in analytical caesium chloride, ultracentrifugation. Electron and fluorescence microscopy of the dyskinetoplastic strains of T. equiperdum suggest that the kDNA network is fragmented and dispersed throughout the mitochondrion. The fragmentation and dispersion of the kDNA, rather than a reduction in the amount of kDNA, is the cause of the lack of kinetoplast staining in the dyskinetoplastic strains of T. equiperdum. Acriflavine-treated cultures of C. fasciculata show a decrease in the amount of kDNA (p = 1.703 g/cm3) corresponding to the percentage of dyskinetoplastic cells in the cultures. Electron and fluorescence microscopy of acriflavine-treated cultures of C. fasciculata show the loss of the kDNA network in cells which lack Giemsa and Feulgen staining, confirming the hypothesis that the kDNA is lost in dyskinetoplastic trypanosomatids from insects. Possible modes of acriflavine action are considered and a proposed mechanism for acriflavine action in trypanosomes from mammals is presented.

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Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei.

The Journal of Cell Biology ◽

10.1083/jcb.76.2.293 ◽

1978 ◽

Vol 76 (2) ◽

pp. 293-309 ◽

Cited By ~ 73

Author(s):

A H Fairlamb ◽

P O Weislogel ◽

J H Hoeijmakers ◽

P Borst

Keyword(s):

Electron Microscopy ◽

Trypanosoma Brucei ◽

Contour Length ◽

Kinetoplast Dna ◽

East African ◽

S1 Nuclease ◽

Isolation And Characterization ◽

Trypanosoma Equiperdum ◽

Double Networks

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.

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