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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide. This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones. The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage. Enhanced DNA cleavage in the presence of N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo. DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II. The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant. Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.
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PMID:A mutation in yeast topoisomerase II that confers hypersensitivity to multiple classes of topoisomerase II poisons. 1071 16

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.
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PMID:The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner. 1109 68

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.
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PMID:Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis. 1109 95

The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.
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PMID:Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. 1145 83

The genes encoding subunits A and B of DNA gyrase and subunits C and E of topoisomerase IV of Listeria monocytogenes, gyrA, gyrB, parC and parE, respectively, were cloned and sequenced. Compared with the sequences of quinolone-susceptible bacteria, such as Escherichia coli and Bacillus subtilis, the quinolone resistance-determining region (QRDR) of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed the substitutions Ser-84-->Thr and Asp/Glu-88-->Phe, two amino acid variations at hot spots, commonly associated with resistance to quinolones. No relevant divergences from QRDR consensus sequences were observed in GyrB or both topoisomerase IV subunits. Thus, it could be argued that the amino acid substitutions in GyrA would explain the intrinsic resistance of L. monocytogenes to nalidixic acid. In order to analyse the actual role of the GyrA alterations, a plasmid-encoded gyrA allele was mutated and transformed into L. monocytogenes. However, these heterodiploid strains were not affected in their resistance to nalidixic acid. The effects of the mutant plasmids on ciprofloxacin and sparfloxacin susceptibility were only modest.
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PMID:Molecular characterization of the genes encoding DNA gyrase and topoisomerase IV of Listeria monocytogenes. 1203 83

We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.
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PMID:Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid. 1207 40

We report that point mutations causing alteration of the fourth alpha-helix (alpha4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser(740)Trp, Gln(743)Pro, and Thr(744)Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca(2+) or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr(744)Ala substitution had minimal effect on Ca(2+)- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the alpha4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser(83)Trp also changed the DNA cleavage pattern generated by Ca(2+) or quinolones. Finally, Thr(744)Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the alpha4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the alpha4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.
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PMID:Importance of the fourth alpha-helix within the CAP homology domain of type II topoisomerase for DNA cleavage site recognition and quinolone action. 1218 23

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 micro g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71-->Val in one, Thr82-->Ile in six, and Ala118-->Thr in one) or in gyrB (six mutations, amino acid changes Asp426-->Asn in five and Arg447-->Leu in one). These changes are similar to those already described in other species except for Asp71-->Val, which is novel, and Ala118-->Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 micro g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.
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PMID:gyrA and gyrB mutations are implicated in cross-resistance to Ciprofloxacin and moxifloxacin in Clostridium difficile. 1238 45

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.
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PMID:Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila. 1497 Jan 88

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.
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PMID:Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species. 1548 21


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