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)

High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.
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PMID:Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine. 283 92

The receptor for epidermal growth factor (EGF) is a single-chain transmembrane polypeptide of relative molecular mass (Mr) 170,000 (170K) which has been implicated in the regulation of both normal and abnormal cell proliferation. It has an externally facing EGF-binding domain and a cytoplasmically facing tyrosine-specific protein kinase site. Although the receptor has been well characterized, the mechanism by which it transmits the growth stimulatory signal from the plasma membrane to the nucleus is unclear. EGF binding to cells has been shown to enhance topoisomerase activity within the cells. Topoisomerases catalyse the interconversion of topological isomers of DNA and thus may influence replication and transcription. Mroczkowski et al. reported that purified EGF receptors of both human and murine origin can nick supercoiled double-stranded (ds) DNA in an ATP-dependent fashion, an activity related to those of topoisomerases. Another related tyrosine kinase, pp60src, has also been reported to have a similar DNA-nicking activity. We have now characterized the EGF receptor-associated DNA-nicking activity by sucrose gradient centrifugation. Our results, presented here, indicate that the DNA-nicking activity is not intrinsic to the EGF receptor, but is found in a distinct molecular species.
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PMID:EGF receptor-associated DNA-nicking activity is due to a Mr-100,000 dissociable protein. 299 1

Many viral oncogenes encode protein-tyrosine kinase activities. However, important in vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to be in vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinh et al. Nature 312: 785-786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylation in vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v-abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylation in vivo.
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PMID:A comparison of topoisomerase I activity in normal and transformed cells. 301 75

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.
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PMID:The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. 302 May 13

Highly purified topoisomerase from Ustilago breaks single-stranded DNA, forming a complex with protein covalently bound to the DNA. Methods used to detect the complexes include a nitrocellulose filter assay, electrophoresis of the DNA-protein complex in agarose gels containing alkali, and isolation of the complex after removal of all but a small oligonucleotide fragment bound to the protein. The linkage of the Ustilago topoisomerase is to the 3' end of the broken strand of DNA. The DNA-protein complex formed is through a phosphodiester bond to tyrosine.
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PMID:Topoisomerase from Ustilago maydis forms a covalent complex with single-stranded DNA through a phosphodiester bond to tyrosine. 302 52

Tyrosine protein kinase activity is associated with at least eight different retrovirus-encoded onc gene products and with cell receptors for epidermal growth factor, platelet-derived growth factor, tumour growth factor and insulin. Both the onc kinases and the growth factor receptors are membrane proteins whose enzymatic activity has been implicated in stimulation of growth. However, the mechanism by which a signal passes from the plasma membrane to the nucleus to initiate growth remains unknown. As DNA topoisomerases catalyse the interconversion of topological isomers of DNA and hence affect DNA replication, transcription and recombination, they may be involved also in stimulation of growth. Several DNA topoisomerases have been shown to form a covalent complex with DNA via a phosphotyrosine linkage. The DNA-protein complex is postulated to be an intermediate in breaking and rejoining of DNA. The aim of the present study was to determine whether tyrosine protein kinases modulate the activity of topoisomerases by phosphorylating the tyrosine residue involved in DNA binding. We report that incubation of Escherichia coli and calf thymus type I DNA topoisomerases with the Rous sarcoma virus transforming gene product, pp60src, and TPK75, a tyrosine protein kinase purified from normal rat liver, results in a 10-fold loss of topoisomerase activity.
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PMID:Virus- and cell-encoded tyrosine protein kinases inactivate DNA topoisomerases in vitro. 609 21

Conditions which result in DNA strand breakage by the rat liver DNA nicking-closing enzyme lead to the covalent attachment of the 3'-end of the broken strand to the enzyme. Treatment of this complex with pancreatic DNase leaves a residue of 17 +/- 8 nucleotide phosphates still attached to the enzyme. Subsequent nuclease P1 treatment removes all but 2 +/- 1 phosphate residues. Using nuclease P1-treated complexes which had been labeled in the DNA with 32P, the stability of the protein-DNA linkage was studied. The linkage is stable to acid, base, neutral and acidic hydroxylamine, and neutral I2. This pattern of stability rules out essentially all of the possible DNA-protein linkages except for a linkage involving a phosphodiester bond to the amino acid tyrosine. After acid hydrolysis of the 32P-labeled complexes, label was found to be associated with O4-phosphotyrosine, providing a direct demonstration that tyrosine is the amino acid to which the end of the DNA chain is attached.
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PMID:DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine. 626 3

Eukaryotic type I DNA topoisomerase provides swivels for removing torsional strain from the DNA helix during transcription and DNA replication. Previously it has been shown that the enzyme is associated with actively transcribed genes and replicating DNA. Using an inactive mutant form of the protein containing phenylalanine instead of tyrosine at position 723, we have investigated the binding properties of the protein as a function of substrate topology. A series of filter binding assays indicated that the protein strongly prefers to bind superhelical over completely relaxed SV40 DNA. The ability of a supercoiled DNA to compete against a relaxed DNA for binding increases as the number of superhelical turns in the DNA increases. Since positively supercoiled DNA is bound with the same preference as negatively supercoiled DNA, we hypothesize that topoisomerase I binds preferentially at the nodes created by the crossing of two duplex helices. The preference for binding superhelical DNA is also exhibited by the conserved core domain (amino acids 175-659) which is missing the active site region located near the C-terminus. These results suggest that this core domain may target the enzyme in vivo to regions of torsionally strained superhelical DNA.
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PMID:Preferential binding of human topoisomerase I to superhelical DNA. 748 29

Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.
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PMID:Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. 749 3

Anthracenyl-amino acid/dipeptides are novel topoisomerase (topo) inhibitors which can be actively cytotoxic in the low microM range. The present studies have been performed to determine whether cells treated with the topo II catalytic inhibitor NU/ICRF 500 (serine derivative) would manifest cytogenetic lesions consistent with its proposed mechanism of enzyme inhibition. Three other compounds were included for comparison: NU/ICRF 505 (tyrosine) which stabilises topo I cleavable complexes, NU/ICRF 602 (gly-gly) a non-cytotoxic catalytic inhibitor of topo I and II and NU/ICRF 502 (alanine) a non-cytotoxic non-topo inhibitor. Chromosomal damage was measured using the micronucleus test. NU/ICRF 500 (7.5-30 microM) induced an increase in CREST negative micronuclei (11-15 per 500 cells) in human lymphocytes (HL) and blocked the traverse of HL through the cell cycle, with cells accumulating in G2/M at 15 microM drug and G1/S at 30 microM drug. NU/ICRF 502 was without effect in the micronucleus test. NU/ICRF 500 and 602 (90-150 microM) caused no block in passage of synchronised metaphase Chinese hamster ovary cells through mitosis whereas NU/ICRF 505 produced a significant delay. DNA measurements of post-mitotic cells revealed that after NU/ICRF 500 treatment nuclei had a 4C DNA content, indicative of a lack of chromosomal segregation. Normal (2C) DNA content was observed with NU/ICRF 505 and 602. Overall, the data for NU/ICRF 500 are consistent with the cytogenetic modifications expected after catalytic inhibition of topo II and suggest that cell death may be mediated, at least in part, through this mechanism.
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PMID:Cytogenetic evaluation of the mechanism of cell death induced by the novel anthracenyl-amino acid topoisomerase II catalytic inhibitor NU/ICRF 500. 756 93


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