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The Journal of Biological Chemistry Dec 2017Resveratrol, a polyphenol found in various plant sources, has gained attention as a possible agent responsible for the purported health benefits of certain foods, such...
Resveratrol, a polyphenol found in various plant sources, has gained attention as a possible agent responsible for the purported health benefits of certain foods, such as red wine. Despite annual multi-million dollar market sales as a nutriceutical, there is little consensus about the physiological roles of resveratrol. One suggested molecular target of resveratrol is eukaryotic topoisomerase II (topo II), an enzyme essential for chromosome segregation and DNA supercoiling homeostasis. Interestingly, resveratrol is chemically similar to ICRF-187, a clinically approved chemotherapeutic that stabilizes an ATP-dependent dimerization interface in topo II to block enzyme activity. Based on this similarity, we hypothesized that resveratrol may antagonize topo II by a similar mechanism. Using a variety of biochemical assays, we find that resveratrol indeed acts through the ICRF-187 binding locus, but that it inhibits topo II by preventing ATPase domain dimerization rather than stabilizing it. This work presents the first comprehensive analysis of the biochemical effects of both ICRF-187 and resveratrol on the human isoforms of topo II, and reveals a new mode for the allosteric regulation of topo II through modulation of ATPase status. Natural polyphenols related to resveratrol that have been shown to impact topo II function may operate in a similar manner.
Topics: Adenosine Triphosphatases; Allosteric Regulation; Amino Acid Substitution; DNA Topoisomerases, Type II; Dexrazoxane; Humans; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Poly-ADP-Ribose Binding Proteins; Protein Interaction Domains and Motifs; Protein Multimerization; Resveratrol; Saccharomyces cerevisiae Proteins; Topoisomerase II Inhibitors
PubMed: 29074616
DOI: 10.1074/jbc.M117.810580 -
ELife Jan 2022DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo...
DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase; however, robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.
Topics: Archaeal Proteins; DNA Topoisomerases, Type II; DNA, Catenated; Methanosarcina; Single Molecule Imaging; Stereoisomerism
PubMed: 35076393
DOI: 10.7554/eLife.67021 -
International Journal of Molecular... Feb 2023Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their...
Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes-the bacterial type II topoisomerases gyrase and topoisomerase IV-may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water-metal ion bridge that has been found to mediate the major point of interaction between topoisomerase IV and topoisomerase IV and gyrase, as well as gyrase, exists in gyrase. This is the first investigation of the water-metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water-metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water-metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility.
Topics: Quinolones; DNA Topoisomerase IV; Escherichia coli; Water; Anti-Bacterial Agents; Metals; Fluoroquinolones; DNA Gyrase; Topoisomerase II Inhibitors; DNA Topoisomerases, Type II
PubMed: 36769202
DOI: 10.3390/ijms24032879 -
FEBS Letters Mar 2020Topoisomerases, the ubiquitous enzymes involved in all DNA processes across the biological world, are targets for various anticancer and antimicrobial agents. In...
Topoisomerases, the ubiquitous enzymes involved in all DNA processes across the biological world, are targets for various anticancer and antimicrobial agents. In Entamoeba histolytica, the causative agent of amebiasis, we found one of seven unexplored putative topoisomerases to be highly upregulated during heat shock and oxidative stress, and also during the late hours of encystation. Further analysis revealed the upregulated enzyme to be a eukaryotic type IIA topoisomerase (TopoII) with demonstrable activity in vitro. This enzyme is localized to newly forming nuclei during encystation. Gene silencing of the TopoII reduces viability and encystation efficiency. Notable susceptibility of Entamoeba TopoII to prokaryotic topoisomerase inhibitors opens up the possibility for exploring this enzyme as a new antiamoebic target.
Topics: Amebicides; DNA Topoisomerases, Type II; Drug Delivery Systems; Entamoeba histolytica; Heat-Shock Response; Oxidative Stress; Protozoan Proteins; Topoisomerase II Inhibitors
PubMed: 31724164
DOI: 10.1002/1873-3468.13677 -
Proceedings of the National Academy of... Jun 1996Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations...
Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.
Topics: Adenosine Triphosphate; DNA Topoisomerases, Type II; DNA, Circular; Humans; Hydrolysis; Microscopy, Electron, Scanning Transmission; Protein Conformation
PubMed: 8650197
DOI: 10.1073/pnas.93.12.5936 -
Nucleic Acids Research Jun 1997The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing... (Comparative Study)
Comparative Study
The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P =0.11) with regions of high topoisomerase II cleavage site frequency. These regions show no correlation with repetitive sequence or A/T or C/G content and some extend over >10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome.
Topics: Animals; Binding Sites; Chromosome Mapping; DNA; DNA Topoisomerases, Type II; Drosophila; Electrophoresis, Polyacrylamide Gel; Nucleic Acid Conformation; Restriction Mapping; Structure-Activity Relationship
PubMed: 9153300
DOI: 10.1093/nar/25.11.2041 -
Proceedings of the National Academy of... Sep 2003Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer...
Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5'-[beta,gamma-imino]-triphosphate-bound yeast topo II ATPase region (ScT2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Antineoplastic Agents; Binding Sites; DNA Topoisomerases, Type II; Models, Molecular; Protein Conformation; Razoxane; Topoisomerase II Inhibitors
PubMed: 12963818
DOI: 10.1073/pnas.1832879100 -
Genes To Cells : Devoted To Molecular &... Jan 2023Steroid hormones induce the transcription of target genes by activating nuclear receptors. Early transcriptional response to various stimuli, including hormones,...
Steroid hormones induce the transcription of target genes by activating nuclear receptors. Early transcriptional response to various stimuli, including hormones, involves the active catalysis of topoisomerase II (TOP2) at transcription regulatory sequences. TOP2 untangles DNAs by transiently generating double-strand breaks (DSBs), where TOP2 covalently binds to DSB ends. When TOP2 fails to rejoin, called "abortive" catalysis, the resulting DSBs are repaired by tyrosyl-DNA phosphodiesterase 2 (TDP2) and non-homologous end-joining (NHEJ). A steroid, cortisol, is the most important glucocorticoid, and dexamethasone (Dex), a synthetic glucocorticoid, is widely used for suppressing inflammation in clinics. We here revealed that clinically relevant concentrations of Dex and physiological concentrations of cortisol efficiently induce DSBs in G phase cells deficient in TDP2 and NHEJ. The DSB induction depends on glucocorticoid receptor (GR) and TOP2. Considering the specific role of TDP2 in removing TOP2 adducts from DSB ends, induced DSBs most likely represent stalled TOP2-DSB complexes. Inhibition of RNA polymerase II suppressed the DSBs formation only modestly in the G phase. We propose that cortisol and Dex frequently generate DSBs through the abortive catalysis of TOP2 at transcriptional regulatory sequences, including promoters or enhancers, where active TOP2 catalysis occurs during early transcriptional response.
Topics: DNA Breaks, Double-Stranded; Transcription Factors; DNA-Binding Proteins; Glucocorticoids; DNA Repair; Nuclear Proteins; Hydrocortisone; Phosphoric Diester Hydrolases; DNA Topoisomerases, Type II; DNA
PubMed: 36415926
DOI: 10.1111/gtc.12993 -
Molecules (Basel, Switzerland) Aug 2022C4 variation of 4'--demethyl-epipodophyllotoxin (DMEP) is an effective approach to optimize the antitumor spectra of this compound class. Accordingly, two series of...
C4 variation of 4'--demethyl-epipodophyllotoxin (DMEP) is an effective approach to optimize the antitumor spectra of this compound class. Accordingly, two series of novel DMEP derivatives were synthesized, and as expected, the antitumor spectra of these derivatives varied with different C4 substituents. Notably, most compounds showed significant inhibition against the etoposide (2)-resistant KBvin cells. Four of the compounds (, , and ) induced protein-linked DNA break (PLDB) levels higher than those of GL-331 () and , and are assumed to be topoisomerase II (topo II) poisons more potent than and . Compound , a potent topo II poison highly effective against KBvin cells, was further evaluated with a panel of tumor cells and was most active against HepG2. This compound also exhibited apparent in vivo antitumor efficacy in hepatoma 22 (H22) mouse model. The results indicated that C4 derivation of DMEP is a feasible approach to identify potent topo II inhibitors with optimized antitumor profiles.
Topics: Animals; Antineoplastic Agents; DNA Topoisomerases, Type II; Drug Screening Assays, Antitumor; Mice; Podophyllotoxin; Structure-Activity Relationship; Topoisomerase II Inhibitors
PubMed: 35956979
DOI: 10.3390/molecules27155029 -
PloS One Feb 2011Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of...
BACKGROUND
Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.
METHODOLOGY/PRINCIPAL FINDINGS
We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.
CONCLUSIONS/SIGNIFICANCE
The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme's K(D) for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.
Topics: Antigens, Neoplasm; DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Fluorescence Polarization; Humans; Ions; Metals; Models, Biological; Mutant Proteins; Osmolar Concentration; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae
PubMed: 21358820
DOI: 10.1371/journal.pone.0014693