topoisomerase ii activity

Inhibition of Topoisomerase II DNA Relaxation Activity. eﬀect on human DNA topoisomerase II relaxation activity was investigated, which highlighted the good correlation between the antiproliferative eﬀect and topoisomerase II inhibition. stereochemistry Tanshinone IIA synergistically enhances the antitumor ... These findings indicate a novel physiological function for topoisomerase II and a mechanism by which the enzyme can selectively remove damaged cells from a population. Topoisomerase II, extracted from nuclei with 0.35 M NaCI, was assayed by the P4 DNA unknotting assay (Fig. Topoisomerase II inhibitors 1 July 2011 Topoisomerase II Inhibitors Topoisomerase II inhibitors are chemicals that inhibit a group of DNA enzymes called type II topoisomerases (topoisomerase IIs). Type I1 enzymes (topoisomerase II), however, catalyze topology changes by reversibly breaking both strands of the DNA double helix, resulting in a linking number change of two (Vosberg 1985; Wang 1987). Topoisomerase I (E. coli In this process, these enzymes change the linking number of circular DNA by ±2. A cell-permeable derivative of podophyllotoxin that acts as a topoisomerase II inhibitor (IC₅₀ = 59.2 µM) has major activity against a number of tumors, including germ cell neoplasms, small cell lung cancer, and malignant lymphoma. J. Mol. They are broadly classified into two categories namely, Type II A and Type IIB. Purpose: Topoisomerase IIα content, topoisomerase II catalytic activity and drug sensitivities to the topoisomerase II inhibitors, doxorubicin and etoposide, were examined in a panel of 14 unselected human lung cancer cell lines in order to determine the relationship between topoisomerase II and drug sensitivities to the topoisomerase II inhibitors. Interlinked DNA nano-circles for measuring … Psorospermin is a natural product that has been shown to have activity against drug-resistant leukemia lines and AIDS-related lymphoma. Doxorubicin (Dox) is the first-line drug for the TNBC treatment, acting as a DNA intercalator and topoisomerase II (Topo II) inhibitor; however, it has been observed to exhibit strong cardiotoxicity. The main difference between Topoisomerase I and II is that topoisomerase I cut one strand of the DNA double helix whereas topoisomerase II cut both strands of the DNA double helix.Furthermore, topoisomerase I do not require ATP hydrolysis while topoisomerase II requires ATP hydrolysis. Structure of a topoisomerase II–DNA–nucleotide complex ... Topoisomerases (or DNA topoisomerases) are enzymes that participate in the overwinding or underwinding of DNA. PIASy-dependent SUMOylation regulates DNA topoisomerase … Kinase Assay. However, the regulatory mechanism of TopoII activity is not understood. Cell 165 , 357–371 (2016). Vanderbilt University Type II topoisomerase - Wikipedia The mechanism is similar in both types of topoisomerase. Targeting topoisomerase II activity in NSCLC with 9 ... In both cell types, novobiocin induced similar reductions in topoisomerase I activity but different reductions in Topo II activity. One of these, heat shock protein-90 (Hsp90), is itself a Read Paper. This interaction disrupts the cleavage-relegation equilibrium of Topo II, … Both representations show only the DNA-binding gate and not the ATPase segment. Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. Reaction products were separated by 1% agarose gel electrophoresis in a TAE (Tris-acetic acid-EDTA) buffer system. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples. Topoisomerase I has also been implicated in knotting and unknotting DNA (1) and in linking complementary rings of single-stranded DNA into double-stranded rings (2). Rather, the activity of the topoisomerase II inhibitors was unexpectedly antagonized. This Paper. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. In humans, … It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. 0 1996 Cold Spring Harbor Laboratory Press 0-87969-459-9/96 $5 + .OO 587. The most potent derivative, 32, was shown to induce the apoptosis pathway. Material and Methods We conclude that the drug-DNA complex formed blocks topoisomerase II binding and activity leading to catalytic inhibition of the enzyme and the induction of apoptosis and programmed cell death. 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. The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. The topoisomerase II activity assay was performed using recombinant human topoisomerase IIα (Top2A) or Top2B, which were purified as described previously (Gilroy and Austin, 2011) and kindly provided by … What is Topoisomerase II? Introduction: Topoisomerase II alpha (Topo IIα) has become one of the extensively exploited targets in chemotherapy due to its role in regulating the topological constraints of DNA during replication and transcription. Topoisomerase II Assay Conditions. Furthermore, we see a 100-fold decrease in the LD50 of etoposide, another topo II inhibitor. (ii) ATP independent relaxation activity was not detected. with protein led us to investigate the involvement of topoisomerase II (topo II) in their formation. Topoisomerase is an enzyme tasked with cutting, or influencing DNA to repair breakage and avoid further complication when necessary. Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands. Decatenation assays are carried out using kinetoplast DNA substrate in a final volume of 20-30 µl in topo II reaction buffer (50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2. The Ecoli topoisomerase I is a holoenzyme with three Zn (II) atoms in the tetracysteine motifs near its carboxy terminus. It is 97 kDa in weight. Topoisomerase I has several unusual features. It does not need ATP hydrolyzing to catalyze the topological rearrangement of DNA. 15, 13111–13122. Because Topo II is important as a decatenase activity in vivo, it has an acute preference for binding to DNA ‘nodes’ or duplex/duplex crossovers, where it … Tanshinone IIA (Tan IIA) has a previously confirmed antitumor activity against breast cancer in addition to its well-known cardioprotective effect. DNA Topoisomerase II Structure. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples. Because topoisomerase II is postulated to play a role in DNA repair (14), it was of interest to compare the topoisomerase II activity of Raji and Raji-HN2 cells. Topoisomerase IIs regulate the structure of DNA and are essential in separating multiple intertwined DNA daughter strands after There are several reports in the literature that implicate reduced topoisomerase II activity in the drug resistance phenotype (19, 31-37). In order to manage the DNA tangles and supercoils, the type II topoisomerase cut both the DNA strands simultaneously. Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain). They are broadly classified into two At last one final time, the enzyme opens up and releases the active site and move to another site. DNA Replication in Eukaryotic Cells. Topoisomerase poisons, in contrast to topoisomerase catalytic inhibitors, often have good anticancer activity. The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection. The drug has activity as monotherapy in recurrent acute myelogenous leukemia (AML) and in platinum-refractory ovarian cancer. ASSAY OF TOPOISOMERASE II ACTIVITY . Show abstract. AB - A series of substituted 9-aminoacridines is evaluated for antiproliferative activity toward pancreatic cancer cells. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. Nuclear extracts from human lung cancer cells treated with either the topo II poison etoposide or TPZ under hypoxic conditions had markedly reduced topo II activity as judged by an inability Also, in topoisomerase II-depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. No autophosphorylation of the topoisomerase was observed. Type II topoisomerases are divided into two subfamilies: IIA and IIB. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Schmidt, B., Osheroff, N. & Berger, J. Incubation was for exactly 12 minutes at 37°C. In our present study, it was found that fractions C4 and C5 could strongly inhibit the relaxation activity of topoisomerase II in a dose-dependent manner (Figures 4(c) and 4(d)), suggesting that inhibition of topoisomerases II relaxation activity may be one of the mechanisms of fraction C4- or C5-induced apoptosis. Human recombinant TOP1 and BRD4 were purified and Levens, D. (2014). Moreover, the three main subclasses of topoisomerase I are Type … They need ATP hydrolyzing for this activity. topoisomerase II and 3.3 ,umol of phosphate incorporated per min per mg of kinase, respectively. Download Download PDF. Etoposide … eLife. II. Topoisomerases are ubiquitous enzymes, found in all living organisms. The obtained results highlight 3-methyl-2-phenyl-1H-indole as a Here, we show that PIASy-mediated small ubiquitin-like modifier DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide. Type II topoisomerase change the linking number of circular DNA by ± 2. Etoposide (VP-16, VP-16213) is a semisynthetic derivative of podophyllotoxin, which inhibits DNA synthesis via topoisomerase II inhibition activity. Topoisomerase Assay. The monocyclic 1,4-benzoquinone, HU-331, the direct oxidation product of cannabidiol, inhibits the catalytic activity of topoisomerase II but without inducing DNA strand breaks or generating free radicals, and unlike many … Structure of a topoisomerase II–DNA–nucleotide complex reveals a new control mechanism for ATPase activity. Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. However, topoisomerase II is unique in its ability to catalyze the decatenation of intact double-stranded DNA, a property that allows the enzyme to separate replicated DNA molecules at mitosis (Nitiss, 2009a; Wang, 2002). DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapeutics to kill cancer cells. The main difference between Topoisomerase I and II is that topoisomerase I cut one strand of the DNA double helix whereas topoisomerase II cut both strands of the DNA double helix.Furthermore, topoisomerase I do not require ATP hydrolysis while topoisomerase II requires ATP hydrolysis. However, when positive supercoiling takes place in catenated plasmid, topoisomerase II activity is directed toward decatenation of the molecules before relaxation. Compounds that inhibit the activity of DNA TOPOISOMERASE II. This increase in doxorubicin sensitivity is associated with a 4.5-fold increase in the amount of topo IIα protein and an increase in topo II activity as measured by DNA decatenating and unknotting activities, as well as cleavable complex formation. Topoisomerase-II activity was analyzed in various human leukemic and lymphoblastoid cell-lines with comparison to normal human peripheral blood lymphocytes. typeII topoisomerase, butits activity is notATP-dependent (9). Purpose : To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action. A human Burkitt lymphoma cell line, Raji-HN2, made 10-fold more resistant to nitrogen mustard (HN2) than the parental Raji cell line, exhibited the following characteristics when compared to the parental Raji cells: (i) decreased HN2-induced DNA interstrand crosslinking; (ii) increased (3-fold) DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] activity; (iii) … Experimental 2.1. Type II topoisomerases are topoisomerases that cut both strands of the DNA helix simultaneously in order to manage DNA tangles and supercoils. In eukaryotes, IIA enzymes catalyze the relaxation of positively or negatively supercoiled DNA, as well as the decatenation and unknotting of DNA helices. isoform However, the regulatory mechanism of TopoII activity is not understood. Jinlin Li, Noemi Nagy, Jiangnan Liu, Soham Gupta, Teresa Frisan, Thomas Hennig, Donald P. Cameron, Laura Baranello, Maria G. Masucci Laura Baranello. Moreover, the three main subclasses of topoisomerase I are Type … Topoisomerase alpha is showns as a monomer and topoisomerase beta is shown as a dimer. DNA knotting probability of chromatin is not minimized by an intrinsic topology-simplifying capacity of Topoisomerase II. Neither Cohesin nor Smc5/6 SMC complexes are required to minimize DNA knotting. Condensin inactivation increases the frequency of DNA knots throughout the cell cycle. Name Topoisomerase II Inhibitors Accession Number DBCAT000549 Description. Topoisomerase II (topo II), which catalyzes a transient breakage and reunion of double-stranded DNA, was the first protein shown to be essential for mitotic chromosome condensation (Uemura et al., 1987).A role for topo II in the structural maintenance of mitotic chromosomes has been suggested on the basis of the finding that topo II is a major constituent … Topoisomerase II activity was high in cervix, colon and lung cancers relative to breast cancer. Full PDF Package Download Full PDF Package. In the present study, andrographolide and its structurally modified analogues were investigated for their inhibitory activities on hTopo II α enzyme. Topoisomerase II activity contained in these nuclear extracts was quantified using a Topo II assay kit obtained from TopoGEN (Port Orange, Fla). Baranello, L. et al. (2002). Like topoisomerase I, topoisomerase II catalyzes relaxation of supercoiled DNA. The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Type II topoisomerases (TOP2s) alter DNA topology and play roles in replication, transcription, recombination, and chromosome condensation and segregation (1, 2).These two-fold symmetric enzymes transiently cleave a pair of opposing phosphodiester bonds four base pairs apart, which generates a TOP2-DNA cleavage complex. The role of DNA topoisomerases (Topo) IIα and IIβ was investigated in various drug-resistant melanoma cells. CAS … The enzymes involved in DNA metabolism may offer targets for chemotherapy, especially in rapidly proliferating cells. Two series of the ferrocenyl and ruthenocenyl analogues of etoposide bearing 1,2,3-triazolyl or aminoalkyl linker were synthesized and evaluated for their cytotoxic properties, influence on the cell cycle, ability to induce tubulin polymerization, and inhibition of topoisomerase II activity. 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