Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

To elucidate yeast chromosome structure and behavior, we examined the breakage of entangled chromosomes in DNA topoisomerase II mutants by hybridization to chromosomal DNA resolved by pulsed-field gel electrophoresis. Our study reveals that large and small chromosomes differ in the nature and distribution of their intertwinings. Probes to large chromosomes (450 kb or larger) detect chromosome breakage, but probes to small chromosomes (380 kb or smaller) reveal no breakage products. Examination of chromosomes with one small arm and one large arm suggests that the two arms behave independently. The acrocentric chromosome XIV breaks only on the long arm, and its preferred region of breakage is approximately 200 kb from the centromere. When the centromere of chromosome XIV is relocated, the preferred region of breakage shifts accordingly. These results suggest that large chromosomes break because they have long arms and small chromosomes do not break because they have small arms. Indeed, a small metacentric chromosome can be made to break if it is rearranged to form a telocentric chromosome with one long arm or a ring with an "infinitely" long arm. These results suggest a model of chromosomal intertwining in which the length of the chromosome arm prevents intertwinings from passively resolving off the end of the arm during chromosome segregation.

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DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.159 ◽

1989 ◽

Vol 9 (1) ◽

pp. 159-168 ◽

Cited By ~ 192

Author(s):

C Holm ◽

T Stearns ◽

D Botstein

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Generation Time ◽

Topoisomerase Ii ◽

Chromosome Breakage ◽

Restrictive Temperature ◽

Dna Topoisomerase Ii ◽

Dna Topoisomerase ◽

Sister Chromatids ◽

A Minor

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

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DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.159-168.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 159-168

Author(s):

C Holm ◽

T Stearns ◽

D Botstein

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Generation Time ◽

Topoisomerase Ii ◽

Chromosome Breakage ◽

Restrictive Temperature ◽

Dna Topoisomerase Ii ◽

Dna Topoisomerase ◽

Sister Chromatids ◽

A Minor

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

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Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks

Journal of Cell Science ◽

10.1242/jcs.105.2.563 ◽

1993 ◽

Vol 105 (2) ◽

pp. 563-569 ◽

Cited By ~ 8

Author(s):

D.J. Clarke ◽

R.T. Johnson ◽

C.S. Downes

Keyword(s):

Chromosome Segregation ◽

Mammalian Cells ◽

Topoisomerase Ii ◽

Dna Strand Breaks ◽

Temperature Sensitive ◽

Dna Topoisomerase ◽

Strand Breaks ◽

Temperature Sensitive Mutants ◽

Chromatid Segregation ◽

Dna Strand

Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation. In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects. Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g. ionizing radiation) are known to affect progression into and through mitosis. Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide. We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.

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Inhibitors of topoisomerases do not block the passage of human lymphocyte chromosomes through mitosis

Journal of Cell Science ◽

10.1242/jcs.103.1.105 ◽

1992 ◽

Vol 103 (1) ◽

pp. 105-115

Author(s):

A.T. Sumner

Keyword(s):

Chromosome Segregation ◽

Human Lymphocyte ◽

Topoisomerase I ◽

Topoisomerase Ii ◽

Human Lymphocytes ◽

Chromosome Breakage ◽

Chromosome Condensation ◽

Complete Fusion ◽

Topoisomerase Inhibitors ◽

Topoisomerase Ii Inhibitors

Cultured human lymphocytes have been treated with a number of topoisomerase inhibitors, to see whether topoisomerase II is involved in the process of chromosome segregation at anaphase. Results were assessed by examination of cytogenetical preparations of spread chromosomes. Four effects were observed, although no inhibitor produced all four effects. These effects were: inhibition of entry into mitosis; chromosome breakage and rearrangement; inhibition of chromosome condensation; and inhibition of chromosome segregation. Evidence for the last was ambiguous. Although there was evidence that separation of chromatids was affected when cells were treated with colchicine as well as topoisomerase II inhibitors (most notably with nalidixic acid, which resulted in complete fusion of the chromatids), no evidence was obtained to show that, in the absence of colchicine, cells treated with inhibitors could not proceed through anaphase normally. The topoisomerase I inhibitor, camptothecin, differed from the topoisomerase II inhibitors in not showing any effect on chromosome condensation or any significant effect on segregation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6962-6974.1994 ◽

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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A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis

The Journal of Cell Biology ◽

10.1083/jcb.201511080 ◽

2016 ◽

Vol 213 (6) ◽

pp. 651-664 ◽

Cited By ~ 22

Author(s):

Heather Edgerton ◽

Marnie Johansson ◽

Daniel Keifenheim ◽

Soumya Mukherjee ◽

Jeremy M. Chacón ◽

...

Keyword(s):

Catalytic Activity ◽

Binding Site ◽

Chromosome Segregation ◽

Topoisomerase Ii ◽

Histone H3 ◽

Aurora B ◽

Topoisomerase Iiα ◽

Dna Topoisomerase ◽

Topo Ii ◽

Dna Topoisomerase Iiα

Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin–histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6962 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6962-6974 ◽

Cited By ~ 34

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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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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