-
Molecular Biology and Evolution Nov 2022Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically...
Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.
Topics: Phylogeny; Amino Acid Sequence; DNA Topoisomerases, Type II; Archaeal Proteins; Meiosis; Eukaryota
PubMed: 36256608
DOI: 10.1093/molbev/msac227 -
Nature Jul 2014Spatial patterning is a ubiquitous feature of biological systems. Meiotic crossovers provide an interesting example, defined by the classic phenomenon of crossover...
Spatial patterning is a ubiquitous feature of biological systems. Meiotic crossovers provide an interesting example, defined by the classic phenomenon of crossover interference. Here we identify a molecular pathway for interference by analysing crossover patterns in budding yeast. Topoisomerase II plays a central role, thus identifying a new function for this critical molecule. SUMOylation (of topoisomerase II and axis component Red1) and ubiquitin-mediated removal of SUMOylated proteins are also required. The findings support the hypothesis that crossover interference involves accumulation, relief and redistribution of mechanical stress along the protein/DNA meshwork of meiotic chromosome axes, with topoisomerase II required to adjust spatial relationships among DNA segments.
Topics: Chromosomes, Fungal; Crossing Over, Genetic; DNA Topoisomerases, Type II; Meiosis; Mutation; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation
PubMed: 25043020
DOI: 10.1038/nature13442 -
Archives of Toxicology Jun 2018Dronedarone is used to treat patients with cardiac arrhythmias and has been reported to be associated with liver injury. Our previous mechanistic work demonstrated that...
Dronedarone is used to treat patients with cardiac arrhythmias and has been reported to be associated with liver injury. Our previous mechanistic work demonstrated that DNA damage-induced apoptosis contributes to the cytotoxicity of dronedarone. In this study, we examined further the underlying mechanisms and found that after a 24-h treatment of HepG2 cells, dronedarone caused cytotoxicity, G1-phase cell cycle arrest, suppression of topoisomerase II, and DNA damage in a concentration-dependent manner. We also investigated the role of cytochrome P450s (CYPs)-mediated metabolism in the dronedarone-induced toxicity using our previously established HepG2 cell lines expressing individually 14 human CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrated that CYP3A4, 3A5, and 2D6 were the major enzymes that metabolize dronedarone, and that CYP3A7, 2E1, 2C19, 2C18, 1A1, and 2B6 also metabolize dronedarone, but to a lesser extent. Our data showed that the cytotoxicity of dronedarone was decreased in CYP3A4-, 3A5-, or 2D6-overexpressing cells compared to the control HepG2 cells, indicating that the parent dronedarone has higher potency than the metabolites to induce cytotoxicity in these cells. In contrast, cytotoxicity was increased in CYP1A1-overexpressing cells, demonstrating that CYP1A1 exerts an opposite effect in dronedarone's toxicity, comparing to CYP3A4, 3A5, or 2D6. We also studied the involvement of topoisomerase II in dronedarone-induced toxicity, and demonstrated that the overexpression of topoisomerase II caused an increase in cell viability and a decrease in γ-H2A.X induction, suggesting that suppression of topoisomerase II may be one of the mechanisms involved in dronedarone-induced liver toxicity.
Topics: Cell Culture Techniques; Cell Cycle; Cell Survival; Cytochrome P-450 Enzyme System; DNA Damage; DNA Topoisomerases, Type II; Dronedarone; Hep G2 Cells; Histones; Humans; Liver
PubMed: 29616291
DOI: 10.1007/s00204-018-2196-x -
The Journal of Biological Chemistry Aug 1983A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate...
A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.
Topics: Animals; DNA Topoisomerases, Type II; Drosophila melanogaster; Electrophoresis, Polyacrylamide Gel; Macromolecular Substances; Molecular Weight
PubMed: 6308010
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1993Immunoprecipitation of DNA topoisomerase II from yeast results in a preparation that contains casein kinase II; this suggests that the two proteins may associate in the...
Immunoprecipitation of DNA topoisomerase II from yeast results in a preparation that contains casein kinase II; this suggests that the two proteins may associate in the intact cell. Purified recombinant topoisomerase II and casein kinase II associate to form a complex in vitro which is stable after topoisomerase II becomes phosphorylated by the kinase. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit alone can efficiently phosphorylate topoisomerase II, the formation of a stable topoisomerase II-casein kinase II association requires the presence of the beta subunit of the kinase. Both proteins engaged in this complex retain their catalytic activities. Naturally occurring polyamines and polyanionic compounds appear to be crucial factors governing the interaction between the two proteins. Although the biological significance of a stable catalytically active topoisomerase II-casein kinase II molecular complex remains to be defined, these observations suggest the possibility of a novel mechanism regulating topoisomerase II and casein kinase II activities.
Topics: Amino Acid Sequence; Animals; Casein Kinase II; DNA Topoisomerases, Type II; DNA, Viral; Heparin; Heparitin Sulfate; Kinetics; Macromolecular Substances; Molecular Sequence Data; Moths; Phosphorylation; Protein Serine-Threonine Kinases; Recombinant Proteins; Simian virus 40; Spermine; Substrate Specificity; Suramin; Transfection
PubMed: 8226802
DOI: No ID Found -
The Biochemical Journal Jul 1993Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster...
Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.
Topics: Animals; Blotting, Western; CHO Cells; Cell Cycle; Cricetinae; Cricetulus; DNA Topoisomerases, Type II; Phosphorylation; Precipitin Tests
PubMed: 8392338
DOI: 10.1042/bj2930297 -
PLoS Biology Aug 2008Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can...
Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II-depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II-depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule-kinetochore attachments by allowing proper activation of Aurora B kinase.
Topics: Animals; Aurora Kinase B; Aurora Kinases; Cell Cycle Proteins; Cells, Cultured; Centromere; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila Proteins; Drosophila melanogaster; Enzyme Activation; HeLa Cells; Humans; Kinetochores; Microtubules; Mitosis; Protein Serine-Threonine Kinases; RNA Interference; Sister Chromatid Exchange; Spindle Apparatus; Topoisomerase II Inhibitors
PubMed: 18752348
DOI: 10.1371/journal.pbio.0060207 -
Nature Reviews. Cancer May 2009Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and... (Review)
Review
Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.
Topics: Animals; Antineoplastic Agents; Catalysis; DNA; DNA Repair; DNA Topoisomerases, Type II; Enzyme Inhibitors; Humans; Topoisomerase II Inhibitors
PubMed: 19377506
DOI: 10.1038/nrc2607 -
European Journal of Biochemistry Feb 1985It has recently been suggested that topoisomerases could be important targets for several DNA intercalating drugs used in cancer therapy. This prompted us to purify and...
It has recently been suggested that topoisomerases could be important targets for several DNA intercalating drugs used in cancer therapy. This prompted us to purify and characterize a type II topoisomerase in a highly tumorigenic transplantable rabbit tumor isolated from a skin carcinoma associated with cottontail rabbit papillomavirus. We have found that the decatenating activity present in tumor cells was 40-100 times higher than that in the rabbit liver, while no activity could be found in skin extracts. The type II topoisomerases purified from tumor and liver cells consist of two subunits with molecular masses of about 160 kDa. The conditions of the reactions of relaxation, unknotting and decatenation catalyzed by these topoisomerases II were found to be similar to those observed with enzymes of other eukaryotic cells. In the course of the purification of the VX2 enzyme, we isolated and characterized a protein of about 30 kDa in whose presence the topoisomerase II was able to catenate very efficiently supercoiled DNA molecules. This protein has the same electrophoretic mobility as an H1-2 histone, and cross-reacts with an anti-H1 antiserum. The VX2 topoisomerase II as well as the VX2 tumor should constitute useful models for assays of antitumoral drugs.
Topics: Animals; Collodion; DNA Topoisomerases, Type II; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Histones; Immunochemistry; Liver; Neoplasm Proteins; Neoplasm Transplantation; Papillomaviridae; Rabbits; Skin Neoplasms; Tumor Virus Infections
PubMed: 2982598
DOI: 10.1111/j.1432-1033.1985.tb08677.x -
The EMBO Journal Jun 2006Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their...
Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.
Topics: Chromatin; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Superhelical; Nucleic Acid Conformation; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 16710299
DOI: 10.1038/sj.emboj.7601142