scholarly journals Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

1992 ◽  
Vol 117 (5) ◽  
pp. 921-934 ◽  
Author(s):  
CE Shamu ◽  
AW Murray
Keyword(s):  
Sister Chromatid ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Egg Extracts ◽  
Chromosome Separation ◽  
Sister Chromatid Separation ◽  
Sperm Nuclei ◽  
Cytostatic Factor

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

scholarly journals Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome compaction and individualization

2020 ◽  
Author(s):  
Pavan Choppakatla ◽  
Bastiaan Dekker ◽  
Erin E. Cutts ◽  
Alessandro Vannini ◽  
Job Dekker ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Chromosome Morphology ◽  
Linker Histone ◽  
Dna Topoisomerase Ii ◽  
Dna Amount ◽  
Dna Topoisomerase ◽  
Dna Loop ◽  
Egg Extracts ◽  
Topo Ii

SummaryDNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 regulates chromatin levels of condensins and topo II. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer likely through shortening the average loop size and reducing DNA amount in each layer of mitotic loops. Furthermore, H1.8-mediated suppression of condensins and topo II binding to chromatin limits chromosome individualization by preventing resolution of interchromosomal linkages. While linker histones locally compact DNA by clustering nucleosomes, we propose that H1.8 controls chromosome morphology and topological organization through restricting the loading of condensins and topo II on chromatin.

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

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.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Tannins as Potent Inhibitors of DNA Topoisomerase II In Vitro

1993 ◽  
Vol 82 (5) ◽  
pp. 487-492 ◽  
Author(s):  
Yoshiki Kashiwada ◽  
Gen-Ichiro Nonaka ◽  
Itsuo Nishioka ◽  
Kenneth Jiann-Hung Lee ◽  
Ibrahim Bori ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase

scholarly journals Antimalarial 9-Anilinoacridine Compounds Directed at Hematin

2003 ◽  
Vol 47 (12) ◽  
pp. 3708-3712 ◽  
Author(s):  
Saranya Auparakkitanon ◽  
Wilai Noonpakdee ◽  
Raymond K. Ralph ◽  
William A. Denny ◽  
Prapon Wilairat
Keyword(s):  
Topoisomerase Ii ◽  
Rapid Onset ◽  
Stoichiometric Ratio ◽  
The Novel ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Molar Ratios ◽  
Low Concentrations ◽  
Absorbance Changes

ABSTRACT Antimalarial 9-anilinoacridines are potent inhibitors of parasite DNA topoisomerase II both in vitro and in situ. 3,6-Diamino substitution on the acridine ring greatly improves parasiticidal activity against Plasmodium falciparum by targeting DNA topoisomerase II. A series of 9-anilinoacridines were investigated for their abilities to inhibitβ -hematin formation, to form drug-hematin complexes, and to enhance hematin-induced lysis of red blood cells. Inhibition ofβ -hematin formation was minimal with 3,6-diamino analogs of 9-anilinoacridine and greatest with analogs with a 3,6-diCl substitution together with an electron-donating group in the 1′-anilino position. On the other hand, the presence of a 1′-N(CH3)2 group in the anilino ring produced compounds that strongly inhibited β-hematin formation but which did not appear to be sensitive to the nature of the substitutions in the acridine nucleus. The derivatives bound hematin, and Job's plots of UV-visible absorbance changes in drug-hematin complexes at various molar ratios indicated a stoichiometric ratio of 1:2. The drugs enhanced hematin-induced red blood cell lysis at low concentrations (<4 μM). These studies open up the novel possibility of development of 9-anilinoacridine antimalarials that target not only DNA topoisomerase II but alsoβ -hematin formation, which should help delay the rapid onset of resistance to drugs acting at only a single site.

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain

1994 ◽  
Vol 14 (10) ◽  
pp. 6962-6974
Author(s):  
Y S Vassetzky ◽  
Q Dang ◽  
P Benedetti ◽  
S M Gasser
Keyword(s):  
Chromosome Segregation ◽  
Protein Interactions ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Protein Protein Interactions ◽  
Dna Topoisomerase ◽  
Terminal Domain ◽  
Terminal Amino ◽  
In Vitro Effects

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

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