Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.
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PMID:Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II. 283 60

Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome.
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PMID:Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells. 283 4

Sites of an endogenous activity that has the properties of a DNA topoisomerase I have been identified on the palindromic ribosomal RNA genes of the slime mould Dictyostelium discoideum. This was done in vitro, by treating isolated nuclei with sodium dodecyl sulphate, which denatures topoisomerase during its cycle of nicking, strand passing and resealing, and hence reveals the DNA cleavages. It was also done in vivo using the drug camptothecin, which is believed to stabilize the cleavable complex of topoisomerase I plus DNA, hence increasing the chances of cleavage when sodium dodecyl sulphate is subsequently added. The cleavages in vitro and in vivo were mapped by indirect end-labelling. Both treatments cause what appear to be strong double-stranded cleavages at 200 and 2200 base-pairs and at 17 X 10(3) base-pairs upstream from the rRNA transcription start. The cleavage at 200 base-pairs was analysed in greater detail using RNA hybridization probes specific for single DNA strands. The cleavage is in fact composed of three closely spaced nicks on each DNA strand. The DNA sequence at each of the nicks is strongly homologous across 15 base-pairs. Sodium dodecyl sulphate-induced cleavage by eukaryotic topoisomerase I is known to yield enzyme covalently attached to the 3' cut end of the DNA. We show that protein-linked DNA restriction fragments with their 3' ends at the cleavage sites are selectively retarded on denaturing gels, which provides strong evidence that the unusual cluster of cleavages is caused by a topoisomerase I. Additionally, the camptothecin results revealed cleavages not only at the specific upstream sites, but also across the transcribed region. Interestingly, the zone of camptothecin-assisted cleavage does not extend as far at the 3' end of the gene as the zone of endogenous nuclease sensitivity.
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PMID:Topoisomerase I cleavage sites identified and mapped in the chromatin of Dictyostelium ribosomal RNA genes. 283 75

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.
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PMID:Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro. 283 26

The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug.
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PMID:DNA topoisomerase-targeting antitumor drugs can be studied in yeast. 284 9

In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisomerase I purified from the wild-type and the mutant cells showed that the enzyme from the CptR cells was markedly resistant to camptothecin as assayed by the drug's effects either on relaxation of supercoiled DNA or on stabilization of the covalent enzyme-DNA intermediate. The presence of a camptothecin-resistant form of DNA topoisomerase I in the mutant cells provides further evidence that this enzyme is the cellular target of camptothecin. Cell hybridization studies between the CptR and CptS cells showed that the hybrids formed between these two cell lines were sensitive to camptothecin. The recessive behavior of the CptR mutation provides a plausible explanation for the reduced topoisomerase I content (about one-half of wild-type cells) of the mutant cells and also for their enhanced sensitivity towards inhibitors of topoisomerase II.
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PMID:Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I. 284 51

The supercoiling of 2 micron DNA in yeast by a process or processes that generate positively and negatively supercoiled domains was shown by the use of yeast DNA topoisomerase mutants expressing Escherichia coli DNA topoisomerase I, an enzyme that relaxes negative supercoils specifically. Intracellular 2 micron DNA becomes positively supercoiled in yeast top1 top2 ts strains expressing the E. coli enzyme when neither one of the yeast DNA topoisomerases I and II is functional. Examination of the linking number distributions of plasmids bearing the inducible promoters of GAL1 and GAL10 genes indicates that the generation of supercoiled domains of opposite signs is related to transcription.
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PMID:Supercoiling of intracellular DNA can occur in eukaryotic cells. 284 73

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are both sensitive to camptothecin, an inhibitor of DNA topoisomerase I. An S. cerevisiae DNA repair mutant, rad52, is hypersensitive to the drug. In both species, topoisomerase I mutants totally lacking the enzyme are completely resistant to the drug. A strain with a mutation leading to a temperature-sensitive topoisomerase I exhibits temperature dependence in its in vivo response to camptothecin. A strain carrying a plasmid that overproduces topoisomerase I is hypersensitive to the drug. The rad52 mutant is killed by overproduction of the enzyme, even in the absence of the drug. The response of several of these strains to camptothecin analogs, to DNA topoisomerase II inhibitors, and to other drugs is reported. The cytotoxic effects of camptothecin are discussed in terms of the drug extending the lifetime of a topoisomerase I-DNA covalent intermediate, which is recognized as DNA damage by a DNA repair system.
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PMID:Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. 284 43

RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110,000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH4)2SO4. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes.
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PMID:Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. 285 18

A transposon Tn10 insertion in topA, the structural gene of Escherichia coli DNA topoisomerase I, behaves as an excluded marker in genetic crosses with many strains of E. coli. However, derivative strains that accept this mutant topA allele are readily selected. We show that many of these topA mutant strains contain additional mutations that compensate for the loss of DNA topoisomerase I. Genetic methods for mapping and manipulating such compensatory mutations are described. These methods include a plate-mating test for the ability of strains to accept a topA::Tn10 allele and a powerful indirect selection for transferring compensatory mutations from male strains into non-compensatory female strains. One collection of spontaneous compensatory mutants is analyzed in detail and is shown to include compensatory mutations at three distinct loci: gyrA and gyrB, the genes that encode the subunits of DNA gyrase, and a previously unidentified locus near tolC. Mutations at this third locus, referred to as toc (topoisomerase one compensatory) mutations, do not behave as point mutations in transductional crosses and do not result in lowered DNA gyrase activity. These results show that wild-type strains of E. coli require DNA topoisomerase I, and at least one class of compensatory mutations can relieve this requirement by a mechanism other than reduction of DNA gyrase activity.
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PMID:Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. 298 84


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