10 results on '"Froelich-Ammon, S J"'
Search Results
2. Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation.
- Author
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Shapiro PS, Whalen AM, Tolwinski NS, Wilsbacher J, Froelich-Ammon SJ, Garcia M, Osheroff N, and Ahn NG
- Subjects
- Animals, Antigens, Neoplasm, Cell Line, Cell Nucleus enzymology, Chromatin genetics, DNA, Superhelical metabolism, DNA-Binding Proteins, Dimerization, Drosophila enzymology, Enzyme Activation, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinase 1, Mutation genetics, Phosphoproteins metabolism, Phosphorylation, Precipitin Tests, Protein Binding, Protein Serine-Threonine Kinases genetics, Protein-Tyrosine Kinases genetics, Transfection, Calcium-Calmodulin-Dependent Protein Kinases metabolism, DNA Topoisomerases, Type II metabolism, Isoenzymes metabolism, Mitogen-Activated Protein Kinase Kinases
- Abstract
The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.
- Published
- 1999
- Full Text
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3. Purification of DNA topoisomerase II from Drosophila melanogaster.
- Author
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Froelich-Ammon SJ, Kingma PS, and Osheroff N
- Subjects
- Animals, Cellulose chemistry, Durapatite chemistry, Embryo, Nonmammalian chemistry, Embryo, Nonmammalian metabolism, Cellulose analogs & derivatives, Chromatography, Gel methods, DNA Topoisomerases, Type II isolation & purification, Drosophila melanogaster enzymology, Embryo, Nonmammalian cytology
- Published
- 1999
- Full Text
- View/download PDF
4. Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha.
- Author
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Froelich-Ammon SJ, Dickinson BA, Bevilacqua JM, Schultz SC, and Cech TR
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- Animals, DNA Nucleotidylexotransferase metabolism, DNA, Single-Stranded metabolism, DNA-Directed DNA Polymerase metabolism, Electrophoresis, Agar Gel, Macromolecular Substances, Models, Genetic, Oxytricha genetics, Protein Binding, Protozoan Proteins antagonists & inhibitors, Repetitive Sequences, Nucleic Acid, Species Specificity, Telomerase antagonists & inhibitors, DNA, Protozoan metabolism, DNA-Binding Proteins metabolism, Oxytricha metabolism, Protozoan Proteins metabolism, Telomerase metabolism, Telomere metabolism
- Abstract
Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits alpha and beta on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the alpha subunit. Moreover, the alpha-DNA cross-linked complex was a substrate for telomerase. At higher alpha concentrations, two alpha subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this alpha . DNA . alpha complex may contribute to regulation of telomere length. The alpha . beta . DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of alpha and beta inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.
- Published
- 1998
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5. Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes.
- Author
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Burden DA, Kingma PS, Froelich-Ammon SJ, Bjornsti MA, Patchan MW, Thompson RB, and Osheroff N
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- Animals, Antineoplastic Agents pharmacology, DNA metabolism, Etoposide pharmacology, Hydrolysis, Kinetics, Protein Binding, Antineoplastic Agents metabolism, DNA drug effects, DNA Topoisomerases, Type II metabolism, Drosophila melanogaster enzymology, Etoposide metabolism
- Abstract
Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.
- Published
- 1996
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6. Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase.
- Author
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Froelich-Ammon SJ, Burden DA, Patchan MW, Elsea SH, Thompson RB, and Osheroff N
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- Amino Acid Sequence, Animals, Base Sequence, Binding, Competitive, Chemokines pharmacology, Cloning, Molecular, DNA Primers, Humans, Iodine Radioisotopes, Kinetics, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Receptors, Interleukin-8B, Recombinant Fusion Proteins pharmacology, Recombinant Proteins biosynthesis, Sequence Homology, Amino Acid, Interleukin-8 metabolism, Receptors, Interleukin metabolism, Recombinant Proteins metabolism
- Abstract
Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.
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- 1995
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7. Topoisomerase poisons: harnessing the dark side of enzyme mechanism.
- Author
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Froelich-Ammon SJ and Osheroff N
- Subjects
- Animals, Bacteria enzymology, Cell Death, DNA chemistry, DNA metabolism, DNA Replication, Enzyme Inhibitors toxicity, Topoisomerase I Inhibitors, Topoisomerase II Inhibitors, DNA Topoisomerases, Type I metabolism, DNA Topoisomerases, Type II metabolism, Enzyme Inhibitors pharmacology
- Published
- 1995
- Full Text
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8. Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization of enzyme-drug interactions by fluorescence spectroscopy.
- Author
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Froelich-Ammon SJ, Patchan MW, Osheroff N, and Thompson RB
- Subjects
- Base Sequence, DNA chemistry, DNA Damage, DNA Topoisomerases, Type II chemistry, DNA Topoisomerases, Type II isolation & purification, Ellipticines chemistry, Ellipticines toxicity, Hydrogen-Ion Concentration, Kinetics, Models, Structural, Molecular Sequence Data, Oligodeoxyribonucleotides metabolism, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Saccharomyces cerevisiae drug effects, Spectrophotometry, DNA metabolism, DNA Topoisomerases, Type II metabolism, Ellipticines metabolism, Saccharomyces cerevisiae enzymology
- Abstract
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.
- Published
- 1995
- Full Text
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9. Site-specific cleavage of a DNA hairpin by topoisomerase II. DNA secondary structure as a determinant of enzyme recognition/cleavage.
- Author
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Froelich-Ammon SJ, Gale KC, and Osheroff N
- Subjects
- Animals, Base Sequence, DNA chemistry, Drosophila melanogaster enzymology, Hydrolysis, Molecular Sequence Data, Substrate Specificity, DNA metabolism, DNA Topoisomerases, Type II metabolism, Nucleic Acid Conformation
- Abstract
To further define the nucleic acid determinants that govern the recognition of DNA by topoisomerase II, the ability of the enzyme to cleave a 51-base oligonucleotide that contained a centrally located 19-base hairpin was characterized. Topoisomerase II cleaved the 51-mer in a site-specific fashion, within the hairpin, one nucleotide from the 3'-base of the stem. Protein denaturants were not required to trap cleavage products. Although the sequence of the oligonucleotide influenced levels of enzyme-mediated DNA scission, it did not affect the spatial location of cleavage. DNA scission required a double-stranded/single-stranded junction at the 3'-base of the hairpin and a tail (either single- or double-stranded) at least 8 bases in length on the 5'-side. Cleavage was not observed when base-pairing within the oligonucleotide was eliminated or when the hairpin was extended to produce a completely double-stranded substrate. Finally, the enzyme displayed a size constraint for both the stem and loop structures of the hairpin. These results indicate that topoisomerase II is capable of recognizing secondary structure within nucleic acids and identifies the first secondary structure-specific DNA recognition/cleavage site for the type II enzyme.
- Published
- 1994
10. Novel 1-8-bridged chiral quinolones with activity against topoisomerase II: stereospecificity of the eukaryotic enzyme.
- Author
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Froelich-Ammon SJ, McGuirk PR, Gootz TD, Jefson MR, and Osheroff N
- Subjects
- Animals, Cell Survival drug effects, Cells, Cultured, DNA metabolism, Drosophila melanogaster, Escherichia coli enzymology, Eukaryotic Cells enzymology, Ofloxacin analogs & derivatives, Ofloxacin chemical synthesis, Stereoisomerism, Quinolones pharmacology, Topoisomerase II Inhibitors
- Abstract
A series of novel C-7 quinolyl-substituted enantiomers of ofloxacin were used to determine the stereospecificity of topoisomerase II for the C-11 methyl group in tricyclic quinolones. In all cases, the S isomer was the most active compound against the eukaryotic enzyme. It was approximately 2.2-fold more potent than the R isomer at inhibiting the overall catalytic activity of topoisomerase II (as monitored by DNA relaxation assays). A markedly greater difference in quinolone activity was observed in enzyme-mediated DNA cleavage reactions. While the S enantiomer stimulated nucleic acid breakage approximately 3.5-fold, the R compound did not enhance and, in fact, decreased initial DNA cleavage levels by approximately 50%. The activity of the racemic mixture more closely resembled that of the R enantiomer. In competition experiments, the DNA cleavage-enhancing effects of the S isomer were attenuated by the R compound. Taken together, these latter results indicate that the R enantiomer is an antagonist of S isomer-promoted topoisomerase II-mediated DNA cleavage. Finally, the cytotoxic potential of quinolyl-substituted ofloxacin analogs correlated with the ability to stimulate enzyme-mediated DNA cleavage. Thus, stereochemistry appears to be a governing factor for the potential development of tricyclic quinolones as topoisomerase II-targeted drugs with antineoplastic activity.
- Published
- 1993
- Full Text
- View/download PDF
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