scholarly journals A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation

2013 ◽  
Vol 203 (3) ◽  
pp. 471-486 ◽  
Author(s):  
Andrew B. Lane ◽  
Juan F. Giménez-Abián ◽  
Duncan J. Clarke
Keyword(s):  
Posttranslational Modifications ◽  
Mitotic Chromosome ◽  
Histone H3 ◽  
Important Class ◽  
Histone Tail ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Chromosome Formation ◽  
Dna Topoisomerase Iiα

DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

scholarly journals Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

2004 ◽  
Vol 15 (12) ◽  
pp. 5700-5711 ◽  
Author(s):  
Adam J. Carpenter ◽  
Andrew C.G. Porter
Keyword(s):  
Cell Line ◽  
Human Cell ◽  
Fluorescent Protein ◽  
Human Cell Line ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Green Fluorescent ◽  
Dna Topoisomerase Iiα

DNA Topoisomerase IIα (topoIIα) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIα expression can be silenced in all cells by the addition of tetracycline. TopoIIα-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIα fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIα as an indicator for endogenous topoIIα dynamics, our analyses provide new evidence that topoIIα plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIβ and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

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

2016 ◽  
Vol 213 (6) ◽  
pp. 651-664 ◽  
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.

scholarly journals PIASy-dependent SUMOylation regulates DNA topoisomerase IIα activity

2010 ◽  
Vol 191 (4) ◽  
pp. 783-794 ◽  
Author(s):  
Hyunju Ryu ◽  
Maiko Furuta ◽  
Donald Kirkpatrick ◽  
Steven P. Gygi ◽  
Yoshiaki Azuma
Keyword(s):  
Dna Cleavage ◽  
Biochemical Analysis ◽  
Regulatory Mechanism ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Centromeric Dna ◽  
Anaphase Onset ◽  
Dependent Inhibition ◽  
Dna Topoisomerase Iiα

DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.

scholarly journals Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

2012 ◽  
Vol 199 (5) ◽  
pp. 755-770 ◽  
Author(s):  
Kumiko Samejima ◽  
Itaru Samejima ◽  
Paola Vagnarelli ◽  
Hiromi Ogawa ◽  
Giulia Vargiu ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Intrinsic Structure ◽  
Vitro Assay ◽  
Sister Chromatids ◽  
Parallel Pathway ◽  
Chromosome Formation

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic “X” shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the “intrinsic structure” of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.

scholarly journals SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

2016 ◽  
Vol 213 (6) ◽  
pp. 665-678 ◽  
Author(s):  
Makoto M. Yoshida ◽  
Lily Ting ◽  
Steven P. Gygi ◽  
Yoshiaki Azuma
Keyword(s):  
Topoisomerase Ii ◽  
Histone H3 ◽  
Direct Interaction ◽  
Binding Activity ◽  
Cell Cycle Checkpoints ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
H3 Phosphorylation ◽  
Chromosomal Passenger ◽  
Dna Topoisomerase Iiα

DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.

scholarly journals Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIα gene promoter

1999 ◽  
Vol 344 (2) ◽  
pp. 367-374 ◽  
Author(s):  
Jeong Ho YOON ◽  
Jeong Kee KIM ◽  
Geun Bae RHA ◽  
Misook OH ◽  
Se-Ho PARK ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Promoter Activity ◽  
Expression System ◽  
Critical Role ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Specific Regulation ◽  
Dna Topoisomerase Iiα

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIα is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5′ and/or 3′ serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIα. The luciferase activities of the mutated reporter plasmids in G0-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIα in growing cells.

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