Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation

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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Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6794 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6794-6803 ◽

Cited By ~ 31

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.

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In vivo inhibition of trypanosome mitochondrial topoisomerase II: effects on kinetoplast DNA maxicircles.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5891 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5891-5897 ◽

Cited By ~ 17

Author(s):

T A Shapiro ◽

A F Showalter

Keyword(s):

Topoisomerase Ii ◽

Potent Inhibitor ◽

Complex Structure ◽

Sedimentation Coefficient ◽

Cleavage Product ◽

Unit Size ◽

Kinetoplast Dna ◽

Etoposide Treatment ◽

Minicircle Dna

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a topologically complex structure composed of interlocked minicircles and maxicircles. We previously reported that etoposide, a potent inhibitor of topoisomerase II, promotes the cleavage of about 20% of network minicircle DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). We now find that virtually all maxicircles are released from kinetoplast DNA networks after trypanosomes are treated with etoposide. As expected for a topoisomerase II cleavage product, the linearized maxicircles have protein bound to both 5' ends. After etoposide treatment, the residual minicircle catenanes have a sedimentation coefficient which is only 70% that of controls, and by electron microscopy the networks are less compact. Double-size networks, the characteristic dumbbell-shape forms that normally arise in the final stages of network replication, are replaced by aberrant unit-size forms.

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In vivo inhibition of trypanosome mitochondrial topoisomerase II: effects on kinetoplast DNA maxicircles

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5891-5897.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5891-5897

Author(s):

T A Shapiro ◽

A F Showalter

Keyword(s):

Topoisomerase Ii ◽

Potent Inhibitor ◽

Complex Structure ◽

Sedimentation Coefficient ◽

Cleavage Product ◽

Unit Size ◽

Kinetoplast Dna ◽

Etoposide Treatment ◽

Minicircle Dna

Kinetoplast DNA, the mitochondrial DNA of trypanosomes, is a topologically complex structure composed of interlocked minicircles and maxicircles. We previously reported that etoposide, a potent inhibitor of topoisomerase II, promotes the cleavage of about 20% of network minicircle DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). We now find that virtually all maxicircles are released from kinetoplast DNA networks after trypanosomes are treated with etoposide. As expected for a topoisomerase II cleavage product, the linearized maxicircles have protein bound to both 5' ends. After etoposide treatment, the residual minicircle catenanes have a sedimentation coefficient which is only 70% that of controls, and by electron microscopy the networks are less compact. Double-size networks, the characteristic dumbbell-shape forms that normally arise in the final stages of network replication, are replaced by aberrant unit-size forms.

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Mechanisms of Transcription-Replication Collisions in Bacteria

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.888-895.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 888-895 ◽

Cited By ~ 99

Author(s):

Ekaterina V. Mirkin ◽

Sergei M. Mirkin

Keyword(s):

Escherichia Coli ◽

Direct Contact ◽

Replication Origin ◽

Replication Fork ◽

Mutual Orientation ◽

Physical Interaction ◽

Inducible Promoters ◽

Replication Fork Progression ◽

Replication Intermediates

ABSTRACT While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.

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Maleimide Is a Potent Inhibitor of Topoisomerase II in Vitro and in Vivo: A New Mode of Catalytic Inhibition

Molecular Pharmacology ◽

10.1124/mol.61.5.1235 ◽

2002 ◽

Vol 61 (5) ◽

pp. 1235-1243 ◽

Cited By ~ 34

Author(s):

Lars H. Jensen ◽

Axelle Renodon-Corniere ◽

Irene Wessel ◽

Seppo W. Langer ◽

Birgitte Søkilde ◽

...

Keyword(s):

Topoisomerase Ii ◽

Potent Inhibitor

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Induction of the Bovine Papillomavirus Origin “Onion Skin”-Type DNA Replication at High E1 Protein Concentrations In Vivo

Journal of Virology ◽

10.1128/jvi.76.11.5835-5845.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5835-5845 ◽

Cited By ~ 18

Author(s):

Andres Männik ◽

Kertu Rünkorg ◽

Nele Jaanson ◽

Mart Ustav ◽

Ene Ustav

Keyword(s):

Dna Replication ◽

Skin Type ◽

Bovine Papillomavirus ◽

Two Dimensional Gel Electrophoresis ◽

Reading Frame ◽

E1 Protein ◽

Onion Skin ◽

Origin Region ◽

Replication Intermediates

ABSTRACT We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially “onion skin”-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products.

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The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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