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1.
A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl2. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA. 相似文献
2.
Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated. Under native conditions the enzyme forms a monomer with a relative molar mass of 98.500. A rod-like shape of the molecule was derived from the calculated frictional coefficient. The isoelectric point of the enzyme was determined to be in the range of 7.6–8.0. The enzyme activity is strictly Mg2+ dependent with an optimum at 3 mM Mg2+. The pH optimum ranges within 7.5–9.0. A. eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.Abbreviations DTT
dithiothreitol
- BSA
bovine serum albumin
- EDTA
ethylenediaminetetraacetic acid
- SDS
sodium dodecyl sulfate
- Tris
tris(hydroxymethyl)aminomethane
- PMSF
phenylmethanesulfonyl fluoride
- PAGE
polyacrylamide gel electrophoresis 相似文献
3.
Many viral oncogenes encode protein~yrosine kinase activities. However, importantin vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to bein vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinhet al. Nature
312:785–786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylationin vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v-abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylationin vivo. 相似文献
4.
Pritam Thapa Radha Karki Hoyoung Choi Jae Hun Choi Minho Yun Byeong-Seon Jeong Mi-Ja Jung Jung Min Nam Younghwa Na Won-Jea Cho Youngjoo Kwon Eung-Seok Lee 《Bioorganic & medicinal chemistry》2010,18(6):2245-2254
A series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives were designed, synthesized, and evaluated for their topoisomerase I and II inhibition and cytotoxic activity against several human cancer cell lines. Compounds 10–19 showed moderate topoisomerase I and II inhibitory activity and 20–29 showed significant topoisomerase II inhibitory activity. Structure–activity relationship study revealed that 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl) moiety has an important role in displaying topoisomerase II inhibition. 相似文献
5.
Intracellular distribution of DNA topoisomerase I in fibroblasts from patients with Fanconi's anaemia 总被引:3,自引:0,他引:3
Benhard Auer Hans-Peter Vosberg Ursula Buhre Helmut Klocker Monica Hirsch-Kauffmann Manfred Schweiger 《Human genetics》1982,61(4):369-371
Summary The activity of DNA topoisomerase I (DNA nicking-closing enzyme) was analysed in cytoplasmic and nuclear extracts of six independently derived Fanconi and four normal fibroblast cell lines. In all experiments the total cellular activity was predominantly found in the nuclear extracts (88–100%). In addition, a minor proportion of the enzyme (up to 12%) was randomly present in some of the cytoplasmic fractions of both Fanconi and normal fibroblasts. These results indicate that Fanconi's anaemia is probably not due to or accompanied by a maldistribution of topoisomerase I between nuclei and cytoplasm. 相似文献
6.
Bokun Cheng Thirunavukkarasu Annamalai Shayna Sandhaus Priyanka Bansod Yuk-Ching Tse-Dinh 《PloS one》2015,10(3)
Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development. 相似文献
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8.
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 KD 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. 相似文献9.
An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl2 and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1– extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation. 相似文献
10.
Muzammil Ahmad Yutong Xue Seung Kyu Lee Jennifer L. Martindale Weiping Shen Wen Li Sige Zou Maria Ciaramella Hélène Debat Marc Nadal Fenfei Leng Hongliang Zhang Quan Wang Grace Ee-Lu Siaw Hengyao Niu Yves Pommier Myriam Gorospe Tao-Shih Hsieh Yuk-Ching Tse-Dinh Dongyi Xu Weidong Wang 《Nucleic acids research》2016,44(13):6335-6349
DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals. 相似文献
11.
N. Kunze G. C. Yang Z. Y. Jiang H. Hameister S. Adolph K. -H. Wiedorn A. Richter R. Knippers 《Human genetics》1989,84(1):6-10
Summary Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridzation experiments confirmed these results showing the location of the complete gene on band q11.2–13.1 of chromosome 20, and the location of the pseudogene sequences on band q23–24 of chromosome 1 and q11.2–13.1 of chromosome 22. 相似文献
12.
Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles. 相似文献
13.
Chloroplast DNA topoisomerase I from cauliflower 总被引:1,自引:0,他引:1
An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase. 相似文献
14.
15.
Kyung-Hwa Jeon Han Vit Yu Youngjoo Kwon 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(5):1126-1133
Background
Doxorubicin is commonly using chemotherapeutic agents for breast cancer. However, doxorubicin has limitations in clinical use because of dose-dependent cardiotoxicity and drug resistance. Despite of previously reported studies about mechanisms of doxorubicin resistance including overexpression of P-gp and abnormal expression and mutation of topoisomerase IIα, resistance to this agent still abundantly occur and is regarded as a major obstacle to successful treatment.Methods
We have established doxorubicin resistant T47D cells. Intracellular calcium and ROS levels and calpain activity were measured using fluorometric experiments. Cell viability assay, cell cycle analysis, immunofluorescence and western blot analysis were performed to evaluate m-calpain specific truncation of topoisomerase IIα and molecular mechanism in doxorubicin resistant cells.Results
We observed that doxorubicin treatment increased intracellular calcium and ROS (Reactive Oxygen Species) in parental and doxorubicin resistant T47D cells. The increases in intracellular calcium and ROS were much greater in doxorubicin resistant T47D cells, which led to higher activity of calpains. Hyperactivated m-calpain, but not μ-calpain, specifically induced cleavage of topoisomerase IIα and accumulation of truncated topoisomerase IIα in the cytoplasm. The increase in cytoplasmic truncated topoisomerase IIα reduced the efficacy of doxorubicin. Doxorubicin resistant T47D cells, with hyperactivated m-calpain and truncated cytosolic topoisomerase IIα, obtained cross-resistance to other topoisomerase II-targeting drugs.Conclusion
Hyperactivated m-calpain induced cytoplasmic accumulation of truncated topoisomerase IIα in doxorubicin resistant T47D cells.General significance
These data provide a new mechanism of doxorubicin resistance and suggest a novel strategy for overcoming drug resistance in topoisomerase IIα-targeting therapy. 相似文献16.
《The International journal of biochemistry》1992,24(11):1717-1720
- 1.1. Activity of topoisomerase I and incorporation of [3H]uridine and [14C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun.
- 2.2. A 4-fold transient increase of topoisomerase I activity but not of [3H]uridine or [14C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses.
- 3.3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia.
- 4.4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I.
- 5.5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.
17.
DNA topoisomerase I is a major cellular target for antitumor indolocarbazole derivatives (IND) such as the antibiotic rebeccamycin and the synthetic analogue NB-506 which is undergoing phase I clinical trials. We have investigated the mechanism of topoisomerase I inhibition by a rebeccamycin analogue, R-3, using the wild-type human topoisomerase I and a well-characterized recombinant enzyme, F361S. The catalytic activity of this mutant remains fully intact, but the enzyme is resistant to inhibition by camptothecin (CPT). Here we show that the mutated enzyme is cross-resistant to the rebeccamycin analogue. Despite their profound structural differences, CPT and R-3 interfere similarly with the activity of the wild-type and mutant topoisomerase I enzymes, and the drug-induced cleavable complexes are equally sensitive to the NaCl concentration. CPT and IND likely recognize identical structural elements of the topoisomerase I-DNA covalent complex; however, differences do exist in terms of sequence-specificity of topoisomerase I-mediated DNA cleavage. For the first time, a molecular model showing that CPT and IND share common steric and electronic features is proposed. The model helps to identify a specific pharmacophore for topoisomerase I inhibitors. 相似文献
18.
《Gene》1998,215(2):329-337
Expression of DNA topoisomerase IIα protein varies through the cell cycle with its peak in G2/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIα mRNA stability as well as promoter activity. We isolated the 3′ genomic region of the mouse topoisomerase IIα gene and investigated whether or not the 3′ untranslated region (UTR) of the topoisomerase IIα mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIα 3′ UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3′ region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5′ splice site. The minority of the non-spliced topoisomerase IIα 3′ UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIα 3′ genomic region, or the major spliced form of the 3′ UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIα promoter. Our results strongly suggest that the mouse topoisomerase IIα 3′ UTR by itself is not involved in the cell-cycle-dependent mRNA stability. 相似文献
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20.
《Bioorganic & medicinal chemistry》2014,22(1):211-220
Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα. 相似文献