Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.
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PMID:A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage. 130 70

GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.
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PMID:Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug. 132 74

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.
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PMID:Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro. 168 12

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Streptozotocin (STZ) is a monofunctional nitrosourea employed in the treatment of patients with islet cell tumors. To analyze the role of DNA repair mechanisms in causing resistance to STZ, we evaluated the cytotoxicity by this agent in three human tumor lines that differ with respect to their abilities to repair N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) damaged virus (the Mer phenotype). HT-29, A2182, and BE human tumor lines are high, intermediate and low, respectively, with regard to features that define the Mer phenotype. Our results demonstrated that the order of resistance to STZ is HT-29 greater than A2182 greater than BE. The degree of inhibition of DNA synthesis by STZ was in the following order: BE greater than A2182 greater than HT-29. O6-Alkyltransferase activity was increased markedly in HT-29 cells compared to A2182 cells which, in turn, had significantly increased levels compared to the BE line. Other potential factors such as 3-methyladenine DNA glycosylase activity, the induction by STZ of single-stranded DNA breaks, and the kinetics of repair of these breaks do not clearly underlie differences in cytotoxicity among the three tumor lines. However, increased topoisomerase II activity, as well as enhanced sensitivity to agents that interact with topoisomerase II, was present in A2182 cells compared to BE cells. These findings demonstrate that while O6-alkyltransferase contributes to resistance to STZ in some Mer+ tumor lines, other mechanisms may also contribute to resistance to this agent.
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PMID:Mechanisms underlying resistance to streptozotocin in Mer+ and Mer- human tumor lines. 215 17

The interaction between calf thymus topoisomerase II and DNA has been characterized using a transcription assay. A highly preferred recognition sequence for topoisomerase II was inserted in either direction downstream from a promoter specific for a bacteriophage RNA polymerase. The presence of topoisomerase II-DNA complexes on the template provoked blockage of transcription, yielding RNA transcripts terminated 5' to the topoisomerase II binding site. A footprint of topoisomerase II, derived from transcription towards the complex from either side, revealed that eukaryotic topoisomerase II binds a region of 28 base-pairs with a highly protected central core of 22 base-pairs. The binding region was located symmetrically around the topoisomerase II-mediated cleavage site. In agreement with this result, optimal topoisomerase II-mediated cleavage was observed with a DNA substrate consisting of a 28-mer oligonucleotide homologous to the protected region. Stepwise removal of base-pairs from the ends of the 28-mer gradually reduced the level of enzyme-mediated cleavage.
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PMID:Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting. 217 Jun 62

Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.
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PMID:Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. 255 12

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.
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PMID:Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites. 255 38

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.
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PMID:Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization of enzyme-drug interactions by fluorescence spectroscopy. 779 81

A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.
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PMID:Purification and characterization of a eukaryotic type 1 topoisomerase from pea chloroplast. 810 24


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