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Biophysical Journal Jul 2022Experiments on reconstituted chromosomes have revealed that mitotic chromosomes are assembled even without nucleosomes. When topoisomerase II (topo II) is depleted from...
Experiments on reconstituted chromosomes have revealed that mitotic chromosomes are assembled even without nucleosomes. When topoisomerase II (topo II) is depleted from such reconstituted chromosomes, these chromosomes are not disentangled and form "sparklers," where DNA and linker histone are condensed in the core and condensin is localized at the periphery. To understand the mechanism of the assembly of sparklers, we here take into account the loop extrusion by condensin in an extension of the theory of entangled polymer gels. The loop extrusion stiffens an entangled DNA network because DNA segments in the elastically effective chains are translocated to loops, which are elastically ineffective. Our theory predicts that the loop extrusion by condensin drives the volume phase transition that collapses a swollen entangled DNA gel because the stiffening of the network destabilizes the swollen phase. This may be an important piece to understand the mechanism of the assembly of mitotic chromosomes.
Topics: Chromatin; Chromosomes; DNA; DNA Topoisomerases, Type II; Histones; Mitosis; Nucleosomes
PubMed: 35706364
DOI: 10.1016/j.bpj.2022.06.014 -
Molecular and Cellular Biology Oct 1994We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that...
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.
Topics: Calcium; Chromosomes; Copper; DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Glycerophosphates; Interphase; Microscopy, Electron; Nuclear Proteins; Nucleotides; Peptide Fragments; Phosphorylation; Protein Binding; Protein Conformation; Saccharomyces cerevisiae
PubMed: 7935413
DOI: 10.1128/mcb.14.10.6962-6974.1994 -
International Journal of Oncology Nov 2018The aim of the present study was to determine the role of topoisomerase 1 (TOP1) and topoisomerase 2A (TOP2A) in liver cancer (LC), and to investigate the...
The aim of the present study was to determine the role of topoisomerase 1 (TOP1) and topoisomerase 2A (TOP2A) in liver cancer (LC), and to investigate the inhibitory effect of nitidine chloride (NC) on these two topoisomerases. Immunohistochemistry (IHC) staining and microarray or RNA sequencing data mining showed markedly higher expression of TOP1 and TOP2A at the protein and mRNA levels in LC tissues compared with that in control non-tumor tissues. The prognostic values of TOP1 and TOP2A expression were also estimated based on data from The Cancer Genome Atlas. The elevated expression levels of TOP1 and TOP2A were closely associated with poorer overall survival and disease-free survival rates. When patients with LC were divided into high- and low-risk groups according to their prognostic index, TOP1 and TOP2A were highly expressed in the high-risk group. Bioinformatics analyses conducted on the co-expressed genes of TOP1 and TOP2A revealed that the topoisomerases were involved in several key cancer-related pathways, including the 'p53 pathway', 'pathway in cancer' and 'apoptosis signaling pathway'. Reverse transcription-quantitative polymerase chain reaction and IHC performed on triplicate tumor tissue samples from LC xenografts in control or NC-treated nude mice showed that NC treatment markedly reduced the protein and mRNA expression of TOP1 and TOP2A in LC tissues. Molecular docking studies further confirmed the direct binding of NC to TOP1 and TOP2A. In conclusion, the present findings indicate that TOP1 and TOP2A are oncogenes in LC and could serve as potential biomarkers for the prediction of the prognosis of patients with LC and for identification of high-risk cases, thereby optimizing individual treatment management. More importantly, the findings support TOP1 and TOP2A as potential drug targets of NC for the treatment of LC.
Topics: Animals; Benzophenanthridines; Biomarkers, Tumor; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mice, Inbred BALB C; Molecular Docking Simulation; Molecular Targeted Therapy; Poly-ADP-Ribose Binding Proteins; Topoisomerase II Inhibitors; Xenograft Model Antitumor Assays
PubMed: 30132517
DOI: 10.3892/ijo.2018.4531 -
BMC Developmental Biology Nov 2011Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects...
BACKGROUND
Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue.
RESULTS
Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype.
CONCLUSIONS
We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.
Topics: Animals; Antigens, Neoplasm; Base Sequence; Cell Cycle; Cell Extracts; DNA Topoisomerases, Type II; DNA-Binding Proteins; Diketopiperazines; Embryonic Development; Female; Gene Expression; Gene Knockout Techniques; Genes, Recessive; Isoenzymes; Male; Phenotype; Phylogeny; Piperazines; Point Mutation; Poly-ADP-Ribose Binding Proteins; Sequence Analysis, DNA; Viviparity, Nonmammalian; Xenopus; Zebrafish; Zebrafish Proteins; Zygote
PubMed: 22111588
DOI: 10.1186/1471-213X-11-71 -
Proceedings of the National Academy of... Oct 1988Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by...
Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.
Topics: Base Sequence; Chromosome Banding; Chromosome Mapping; Chromosomes, Human, Pair 17; Cloning, Molecular; DNA; DNA Topoisomerases, Type II; Humans; Molecular Sequence Data
PubMed: 2845399
DOI: 10.1073/pnas.85.19.7177 -
The Journal of Biological Chemistry Jun 1995Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important...
Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.
Topics: Amino Acid Sequence; Amsacrine; DNA; DNA Topoisomerases, Type II; Flavonoids; Humans; Isoenzymes; Molecular Sequence Data; Recombinant Proteins; Saccharomyces cerevisiae
PubMed: 7797575
DOI: 10.1074/jbc.270.26.15739 -
The Journal of Biological Chemistry May 1982An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of...
An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of ATP, has been identified as a type II DNA topoisomerase and is designated yeast DNA topoisomerase II. The formation of the DNA aggregate, which has been shown to be a network of DNA rings that are topologically interlocked, requires the presence of a yeast DNA-binding protein in addition to the topoisomerase. In the absence of the binding protein, yeast DNA topoisomerase II catalyzes decatenation and unknotting of duplex DNA rings and the relaxation of negatively or positively supercoiled DNA. All reactions are ATP-dependent and require Mg(II). Similar to other eukaryotic and phage T4-type II DNA topoisomerases, the yeast enzyme does not catalyze DNA supercoiling under the assay conditions employed. The activity is not sensitive to the gyrase inhibitor nalidixic acid, oxolinic acid, or novobiocin. Coumermycin inhibits the activity, however, at a concentration as low as 5 microgram/ml.
Topics: Adenosine Triphosphate; DNA Topoisomerases, Type II; Kinetics; Microscopy, Electron; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 6279616
DOI: No ID Found -
Nucleus (Austin, Tex.) 2014The double helical structure of DNA lends itself to topological constraints. Many DNA-based processes alter the topological state of DNA, generating torsional stress,... (Review)
Review
The double helical structure of DNA lends itself to topological constraints. Many DNA-based processes alter the topological state of DNA, generating torsional stress, which is efficiently relieved by topoisomerases. Maintaining this topological balance is crucial to cell survival, as excessive torsional strain risks DNA damage. Here, we review the mechanisms that generate and modulate DNA torsion within the cell. In particular, we discuss how transcription-generated torsional stress affects Pol II kinetics and chromatin dynamics, highlighting an emerging role of DNA torsion as a feedback mediator of torsion-generating processes.
Topics: Chromatin; DNA; DNA Topoisomerases, Type II; Humans; Transcription, Genetic
PubMed: 24819949
DOI: 10.4161/nucl.29086 -
Antimicrobial Agents and Chemotherapy Dec 1992Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a...
Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a reaction mixture containing these naphthoquinones and topoisomerase II at an elevated temperature (65 degrees C) resulted in a great reduction in DNA cleavage, suggesting that the mechanism of the topoisomerase II-mediated DNA cleavage induced by these naphthoquinones is through formation of a cleavable complex, as seen with antitumor agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide and demethylepipodophyllotoxin ethylidene-beta-glucoside. Lawson and lapacol, which are structurally related plant metabolites with naphthoquinone moieties, could not induce topoisomerase II-mediated DNA cleavage. Plumbagin and shikonin induced a similar DNA cleavage pattern with topoisomerase II which was different from the cleavage patterns induced with other known topoisomerase II-active drugs. A DNA-unwinding assay with T4 DNA ligase showed that shikonin, lawson, and lapacol did not intercalate into DNA, while plumbagin and 2-methyl-1,4-naphthoquinone intercalate into DNA, but to a lower degree than 4'-(9-acridinylamino)methanesulfon-m-anisidide does.
Topics: Animals; Antineoplastic Agents, Phytogenic; DNA Damage; DNA Topoisomerases, Type II; Drug Synergism; Enzyme Induction; Intercalating Agents; Mice; Mice, Inbred BALB C; Naphthoquinones
PubMed: 1336338
DOI: 10.1128/AAC.36.12.2589 -
Molecular and Cellular Biology Jan 1989The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in...
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.
Topics: Aneuploidy; Chromosome Aberrations; DNA Topoisomerases, Type II; Gene Frequency; Genotype; Hot Temperature; Mitosis; Nondisjunction, Genetic; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 2538717
DOI: 10.1128/mcb.9.1.159-168.1989