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)

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.
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PMID:Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I. 132 12

The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.
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PMID:Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. 796 97

Reverse transcription-PCR-single-strand conformation polymorphism analysis was performed to detect topoisomerase IIalpha mutations using total RNA from 19 bronchial biopsy specimens obtained from 13 patients with small cell lung cancer. An abnormally migrating single-strand conformation polymorphism band was observed in one tumor sample from a patient treated with etoposide-containing chemotherapy. DNA sequence analysis of this tumor showed two transversions at codons 486 (G to A) and 494 (A to G), resulting in two missense mutations (Arg to Lys and Glu to Gly, respectively). The codon 486 mutation was identical to that previously found in two cell lines selected for amsacrine resistance. These results demonstrate that mutations of topoisomerase IIalpha occur in patients with small cell lung cancer. The significance of these mutations in the development of resistance to etoposide needs further investigation.
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PMID:Point mutations of the topoisomerase IIalpha gene in patients with small cell lung cancer treated with etoposide. 864 Aug 4

Fifteen strains of Escherichia coli with MICs of ciprofloxacin (CIP) between 0.015 and 256 micrograms/ml were examined for the presence of mutations in the quinolone resistance-determining region of the gyrA gene and in an analogous region of the parC gene. No mutation was found in a susceptible isolate (MIC of CIP, 0.015 microgram/ml). Four moderately resistant strains (MIC of CIP 0.06 to 4 micrograms/ml) carried one gyrA mutation affecting serine 83, but in only one strain was an additional parC mutation (Gly-78 to Asp) detected. All ten highly resistant strains examined (MIC of CIP, > 4 micrograms/ml) carried two gyrA mutations affecting residues serine 83 and aspartate 87, and at least one parC mutation. These parC mutations included alterations of serine 80 to arginine or isoleucine and glutamate 84 to glycine or lysine. The parC+ and two mutant alleles (parCI-80 and parCI-80,G-84) were inserted into the mobilizable vector pBP507. Transfer of a plasmid-coded parC+ allele into parC+ strains did not alter the susceptibilities towards ciprofloxacin or nalidixic acid, while a significant increase in susceptibility was detectable for parC mutants. This increase, however, did not restore wild-type susceptibility, whereas transfer of a plasmid-coded gyrA+ allele alone or in combination with parC+ did. These data are in agreement with the view that topoisomerase IV is a secondary, less sensitive target for quinolone action in Escherichia coli and that the development of high-level fluoroquinolone resistance in E. coli requires at least one parC mutation in addition to the gyrA mutation(s).
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PMID:Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. 884 44

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.
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PMID:Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene. 885 98

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.
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PMID:Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167. 922 99

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.
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PMID:Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field. 1047 53

Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism.
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PMID:Drop-off during ribosome hopping. 1149 98

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.
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PMID:Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I. 1471 11

Genistein (4'5, 7-trihydroxyisoflavone) occurs as a glycoside (genistin) in the plant family Leguminosae, which includes the soybean (Glycine max). A significant correlation between the serum/plasma level of genistein and the incidence of gender-based cancers in Asian, European and American populations suggests that genistein may reduce the risk of tumor formation. Other evidence includes the mechanism of action of genistein in normal and cancer cells. Genistein inhibits protein tyrosine kinase (PTK), which is involved in phosphorylation of tyrosyl residues of membrane-bound receptors leading to signal transduction, and it inhibits topoisomerase II, which participates in DNA replication, transcription and repair. By blocking the activities of PTK, topoisomerase II and matrix metalloprotein (MMP9) and by down-regulating the expression of about 11 genes, including that of vascular endothelial growth factor (VEGF), genistein can arrest cell growth and proliferation, cell cycle at G2/M, invasion and angiogenesis. Furthermore, genistein can alter the expression of gangliosides and other carbohydrate antigens to facilitate their immune recognition. Genistein acts synergistically with drugs such as tamoxifen, cisplatin, 1,3-bis 2-chloroethyl-1-nitrosourea (BCNU), dexamethasone, daunorubicin and tiazofurin, and with bioflavonoid food supplements such as quercetin, green-tea catechins and black-tea thearubigins. Genistein can augment the efficacy of radiation for breast and prostate carcinomas. Because it increases melanin production and tyrosinase activity, genistein can protect melanocytes of the skin of Caucasians from UV-B radiation-induced melanoma. Genistein-induced antigenic alteration has the potential for improving active specific immunotherapy of melanoma and carcinomas. When conjugated to B43 monoclonal antibody, genistein becomes a tool for passive immunotherapy to target B-lineage leukemias that overexpress the target antigen CD19. Genistein is also conjugated to recombinant EGF to target cancers overexpressing the EGF receptor. Although genistein has many potentially therapeutic actions against cancer, its biphasic bioactivity (inhibitory at high concentrations and activating at low concentrations) requires caution in determining therapeutic doses of genistein alone or in combination with chemotherapy, radiation therapy, and/or immunotherapies. Of the more than 4500 genistein studies in peer-reviewed primary publications, almost one fifth pertain to its antitumor capabilities and more than 400 describe its mechanism of action in normal and malignant human and animal cells, animal models, in vitro experiments, or phase I/II clinical trials. Several biotechnological firms in Japan, Australia and in the United States (e.g., Nutrilite) manufacture genistein as a natural supplement under quality controlled and assured conditions.
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PMID:Anticancer therapeutic potential of soy isoflavone, genistein. 1558 72


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