scholarly journals Mitotic chromosomes are constrained by topoisomerase II–sensitive DNA entanglements

2010 ◽  
Vol 188 (5) ◽  
pp. 653-663 ◽  
Author(s):  
Ryo Kawamura ◽  
Lisa H. Pope ◽  
Morten O. Christensen ◽  
Mingxuan Sun ◽  
Ksenia Terekhova ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Specific Inhibitor ◽  
Elastic Stiffness ◽  
Type I ◽  
Mitotic Chromosomes ◽  
Relaxation Effects ◽  
Double Stranded Dna ◽  
Topo Ii ◽  
Mitotic Chromatin

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II–specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

scholarly journals Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.

1993 ◽  
Vol 120 (3) ◽  
pp. 601-612 ◽  
Author(s):  
T Hirano ◽  
T J Mitchison
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Topo Ii ◽  
Xenopus Egg Extracts

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.

scholarly journals Localization of topoisomerase II in mitotic chromosomes.

1985 ◽  
Vol 100 (5) ◽  
pp. 1716-1725 ◽  
Author(s):  
W C Earnshaw ◽  
M M Heck
Keyword(s):  
Present Article ◽  
Immunoelectron Microscopy ◽  
Polyclonal Antibody ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Experimental Protocol ◽  
Mitotic Chromosomes ◽  
Restricted Domains ◽  
Loop Domains ◽  
Chromosome Scaffold

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.

scholarly journals Contribution of hCAP-D2, a Non-SMC Subunit of Condensin I, to Chromosome and Chromosomal Protein Dynamics during Mitosis

2005 ◽  
Vol 25 (2) ◽  
pp. 740-750 ◽  
Author(s):  
Erwan Watrin ◽  
Vincent Legagneux
Keyword(s):  
Rna Interference ◽  
Mitotic Spindle ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosomal Protein ◽  
Structural Components ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Chromosome Alignment ◽  
Structural Maintenance Of Chromosomes

ABSTRACT Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

scholarly journals SUMO-2/3 regulates topoisomerase II in mitosis

2003 ◽  
Vol 163 (3) ◽  
pp. 477-487 ◽  
Author(s):  
Yoshiaki Azuma ◽  
Alexei Arnaoutov ◽  
Mary Dasso
Keyword(s):  
Topoisomerase Ii ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mitotic Chromosomes ◽  
Protein Inhibition ◽  
Sumo Conjugation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase–anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.

scholarly journals Reciprocal Control of Expression of mRNAs for Osteoclast Differentiation Factor and OPG in Osteogenic Stromal Cells by Genistein: Evidence for the Involvement of Topoisomerase II in Osteoclastogenesis

Endocrinology ◽  
2001 ◽  
Vol 142 (8) ◽  
pp. 3632-3637 ◽  
Author(s):  
Takumi Yamagishi ◽  
Eri Otsuka ◽  
Hiromi Hagiwara
Keyword(s):  
Acid Phosphatase ◽  
Tyrosine Kinases ◽  
Topoisomerase Ii ◽  
Osteoclast Differentiation ◽  
Specific Inhibitor ◽  
Tartrate Resistant Acid Phosphatase ◽  
Spleen Cells ◽  
Differentiation Factor ◽  
Osteoclast Differentiation Factor ◽  
Topo Ii

Abstract Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells with monocyte/macrophage lineage in response to a combination of osteoclast differentiation factor (RANKL) and OPG, a decoy receptor for RANKL, produced by ST2 cells in response to 1α,25-dihydroxyvitamin D3. Treatment of ST2 cells with the natural isoflavonoid genistein for 6 h before coculture with spleen cells inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. When we measured levels of RANKL mRNA in ST2 cells, we found that genistein decreased the level of this mRNA. By contrast, the level of OPG mRNA was enhanced by genistein. Genistein is a specific inhibitor of topoisomerase II (topo II) and an inhibitor of protein tyrosine kinase, as well as being a potent phytoestrogen. To characterize the mode of action of genistein, we examined the effects of an inactive form of genistein (daidzein), 17β-estradiol, inhibitors of topo II, and inhibitors of tyrosine kinases on the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. Among the compounds tested, two inhibitors of topo II, amsacrine and etoposide, attenuated the formation of osteoclast-like cells via reciprocal regulation of the expression of mRNAs for RANKL and OPG in ST2 cells, acting similarly to genistein. Our findings indicate that genistein might inhibit the formation of osteoclast-like cells via inhibition of the activity of topo II, suggesting the novel possibility that topo II might play an important role in osteoclastogenesis.

scholarly journals Cell Cycle-Dependent Control and Roles of DNA Topoisomerase II

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 859 ◽  
Author(s):  
Joyce H. Lee ◽  
James M. Berger
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Topoisomerase Ii ◽  
Type Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Cycle Progression ◽  
Double Stranded Dna ◽  
Topo Ii ◽  
Cycle Dependent

Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.

scholarly journals Guiding functions of the C-terminal domain of topoisomerase IIα advance mitotic chromosome assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Keishi Shintomi ◽  
Tatsuya Hirano
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Fine Tuning ◽  
Terminal Domain ◽  
Nucleosomal Dna ◽  
Egg Extracts ◽  
Guiding Functions ◽  
Topo Ii ◽  
Crowded Environments

AbstractTopoisomerase II (topo II) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood, however, mechanistically how this enzyme regulates this process. In an attempt to further refine the reconstitution assay, we have found that chromosomal binding of Xenopus laevis topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD). Enzymological assays using circular DNA substrates supports the idea that topo IIα first resolves inter-chromatid entanglements to drive individualization and then generates intra-chromatid entanglements to promote thickening. Importantly, only the latter process requires the CTD. By using frog egg extracts, we also show that the CTD contributes to proper formation of nucleosome-depleted chromatids by competing with a linker histone for non-nucleosomal DNA. Our results demonstrate that topo IIα utilizes its CTD to deliver the enzymatic core to crowded environments created during mitotic chromatid assembly, thereby fine-tuning this process.

scholarly journals In vivo dissection of the chromosome condensation machinery

2002 ◽  
Vol 156 (5) ◽  
pp. 805-815 ◽  
Author(s):  
Brigitte D. Lavoie ◽  
Eileen Hogan ◽  
Douglas Koshland
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Histone H3 ◽  
Chromosome Condensation ◽  
Chromatin Binding ◽  
Condensin Complex ◽  
Topo Ii ◽  
Structural Maintenance Of Chromosome

The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented.

scholarly journals CENP-A and topoisomerase-II antagonistically affect chromosome length

2017 ◽  
Vol 216 (9) ◽  
pp. 2645-2655 ◽  
Author(s):  
A.-M. Ladouceur ◽  
Rajesh Ranjan ◽  
Lydia Smith ◽  
Tanner Fadero ◽  
Jennifer Heppert ◽  
...  
Keyword(s):  
Self Assembly ◽  
Topoisomerase Ii ◽  
Chromosome Length ◽  
Chromosome Size ◽  
Mitotic Chromosomes ◽  
Nuclear Trafficking ◽  
C Elegans ◽  
Protein Levels ◽  
Histone H3 Variant ◽  
Topo Ii

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans, CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.

scholarly journals Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

2009 ◽  
Vol 29 (16) ◽  
pp. 4363-4375 ◽  
Author(s):  
Emily A. Outwin ◽  
Anja Irmisch ◽  
Johanne M. Murray ◽  
Matthew J. O'Connell
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Synthetic Lethality ◽  
Mitotic Chromosomes ◽  
Interphase Chromosome ◽  
Chromosome Arm ◽  
Null Mutants

ABSTRACT The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.

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