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

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 Is a Determinant of the Tensile Properties of Yeast Centromeric Chromatin and the Tension Checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0547 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4421-4433 ◽

Cited By ~ 26

Author(s):

Tariq H. Warsi ◽

Michelle S. Navarro ◽

Jeff Bachant

Keyword(s):

Error Correction ◽

Tensile Properties ◽

Mitotic Spindle ◽

Topoisomerase Ii ◽

Dna Topoisomerase Ii ◽

Mechanical Tension ◽

Dna Topoisomerase ◽

Centromeric Chromatin ◽

Sister Chromatids ◽

Sister Kinetochore

Centromeric (CEN) chromatin is placed under mechanical tension and stretches as kinetochores biorient on the mitotic spindle. This deformation could conceivably provide a readout of biorientation to error correction mechanisms that monitor kinetochore–spindle interactions, but whether CEN chromatin acts in a tensiometer capacity is unresolved. Here, we report observations linking yeast Topoisomerase II (Top2) to both CEN mechanics and assessment of interkinetochore tension. First, in top2-4 and sumoylation-resistant top2-SNM mutants CEN chromatin stretches extensively during biorientation, resulting in increased sister kinetochore separation and preanaphase spindle extension. Our data indicate increased CEN stretching corresponds with alterations to CEN topology induced in response to tension. Second, Top2 potentiates aspects of the tension checkpoint. Mutations affecting the Mtw1 kinetochore protein activate Ipl1 kinase to detach kinetochores and induce spindle checkpoint arrest. In mtw1top2-4 and mtw1top2-SNM mutants, however, kinetochores are resistant to detachment and checkpoint arrest is attenuated. For top2-SNM cells, CEN stretching and checkpoint attenuation occur even in the absence of catenation linking sister chromatids. In sum, Top2 seems to play a novel role in CEN compaction that is distinct from decatenation. Perturbations to this function may allow weakened kinetochores to stretch CENs in a manner that mimics tension or evades Ipl1 surveillance.

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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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Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1465 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1465-1476 ◽

Cited By ~ 42

Author(s):

R M Spell ◽

C Holm

Keyword(s):

Chromosome Segregation ◽

Topoisomerase Ii ◽

Acrocentric Chromosome ◽

Chromosome Breakage ◽

Chromosomal Dna ◽

Dna Topoisomerase ◽

Telocentric Chromosome ◽

Small Arms ◽

Yeast Chromosome ◽

And Behavior

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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Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1465-1476.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1465-1476

Author(s):

R M Spell ◽

C Holm

Keyword(s):

Chromosome Segregation ◽

Topoisomerase Ii ◽

Acrocentric Chromosome ◽

Chromosome Breakage ◽

Chromosomal Dna ◽

Dna Topoisomerase ◽

Telocentric Chromosome ◽

Small Arms ◽

Yeast Chromosome ◽

And Behavior

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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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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