scholarly journals Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21

2021 ◽  
Author(s):  
Keiko Morotomi-Yano ◽  
Ken-ichi Yano
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Biological Significance ◽  
Rna Polymerase I ◽  
Atp Depletion ◽  
Dna Topoisomerase Ii ◽  
Nuclear Dynamics ◽  
Dna Topoisomerase ◽  
The Status ◽  
Polymerase I

DNA topoisomerase II (Top2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two Top2 proteins, Top2A and Top2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of Top2B, but the molecular details of subnuclear distributions, particularly of Top2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human Top2A and Top2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the Top2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the Top2 poison etoposide was less effective, indicating the therapeutic relevance of Top2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of Top2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

scholarly journals Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keiko Morotomi-Yano ◽  
Ken-ichi Yano
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Biological Significance ◽  
Rna Polymerase I ◽  
Atp Depletion ◽  
Dna Topoisomerase Ii ◽  
Nuclear Dynamics ◽  
Dna Topoisomerase ◽  
The Status ◽  
Polymerase I

AbstractDNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

scholarly journals Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

Chromosoma ◽  
1988 ◽  
Vol 96 (6) ◽  
pp. 411-416 ◽  
Author(s):  
Kathleen M. Rose ◽  
Jan Szopa ◽  
Fu-Sheng Han ◽  
Yung-Chi Cheng ◽  
Arndt Richter ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Transcription ◽  
Topoisomerase I ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Polymerase I

scholarly journals A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine.

1997 ◽  
Vol 17 (4) ◽  
pp. 1787-1795 ◽  
Author(s):  
O Gadal ◽  
S Mariotte-Labarre ◽  
S Chedin ◽  
E Quemeneur ◽  
C Carles ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Pol Ii ◽  
Pol I ◽  
Polymerase I

A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.

scholarly journals The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning

2020 ◽  
Vol 117 (8) ◽  
pp. 4053-4060 ◽  
Author(s):  
Peter M. Bruno ◽  
Mengrou Lu ◽  
Kady A. Dennis ◽  
Haider Inam ◽  
Connor J. Moore ◽  
...  
Keyword(s):  
Cell Death ◽  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Acquired Resistance ◽  
Clinical Development ◽  
Rna Polymerase I ◽  
Specific Chemical ◽  
Chemical Genetic ◽  
Cellular Processes ◽  
Polymerase I

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes.

scholarly journals Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes.

1990 ◽  
Vol 10 (6) ◽  
pp. 2893-2900 ◽  
Author(s):  
M Dunaway
Keyword(s):  
Rna Polymerase ◽  
Thymidine Kinase ◽  
Topoisomerase Ii ◽  
Xenopus Oocytes ◽  
Initial Period ◽  
Rna Polymerase I ◽  
Time Required ◽  
Polymerase I ◽  
Linear Product

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.

Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes

1990 ◽  
Vol 10 (6) ◽  
pp. 2893-2900
Author(s):  
M Dunaway
Keyword(s):  
Rna Polymerase ◽  
Thymidine Kinase ◽  
Topoisomerase Ii ◽  
Xenopus Oocytes ◽  
Initial Period ◽  
Rna Polymerase I ◽  
Time Required ◽  
Polymerase I ◽  
Linear Product

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.

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.

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