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

---

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

We introduced a series of Pro substitutions within and near the alpha4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase IIalpha, and analyzed if this region is involved in determination of anti-cancer drug sensitivity in a temperature- sensitive yeast strain (top2-4 allele). Among the 19 mutants generated, H759P and N770P showed resistance to etoposide and doxorubicin at the non-permissive temperature, where cell growth depends on activity of the human enzyme. For these residues, mutants with an Ala substitution were further created, in which H759A also showed resistance to etoposide. H759P, H759A and N770P were expressed, purified and subjected to in vitro measurement of drug sensitivity. They generated lower amounts of the etoposide-induced cleavable complexes, and were also found to have lower decatenation activity than the wild-type. In the crystal structure, the yeast equivalent of His759 is found in the vicinity of the Arg713, a putative anchoring residue of the 3'-side of cleaved DNA strands. These results suggest that His759 and the other alpha4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase IIalpha, via interaction with cleaved DNA.
...
PMID:The alpha4 residues of human DNA topoisomerase IIalpha function in enzymatic activity and anticancer drug sensitivity. 1502 36

Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, alpha-amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor-1 knockout mice were resistant to CPT/TNF-induced apoptosis. CPT treatment completely abrogated the TNF-induced NF-kappa B activation, and mRNA expression of the antiapoptotic factors TNF-receptor associated factor 2, FLICE-inhibitory protein, and X-linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (zDEVD-fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF-induced apoptosis. In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.
...
PMID:Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis. 1512 60

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.
...
PMID:DNA ligation catalyzed by human topoisomerase II alpha. 1549 Nov 48

A survey of emm gene sequences and an analysis of the pulsed-field electrophoretic profiles of 30 Streptococcus pyogenes isolates with reduced susceptibilities to ciprofloxacin detected the prevalence of isolates with emm type 6 and considerable genetic diversity among isolates. The mechanism of ciprofloxacin resistance in these isolates was based on point mutations in topoisomerase IV subunit C encoded by parC, mainly replacement of serine-79 by alanine.
...
PMID:Streptococcus pyogenes pharyngeal isolates with reduced susceptibility to ciprofloxacin in Spain: mechanisms of resistance and clonal diversity. 1561 24

We report the synthesis of an original series of oxoazabenzo[de]anthracenes conjugated to an amino acid: Ala, Phe, Pro, Lys, or Gly (4a-e, respectively). The compounds, derived from 1,8-dihydroxyanthracene-9,10-dione, were studied for DNA binding and cytotoxicity. Melting temperature, fluorescence quenching, and surface plasmon resonance methods all indicated that the lysine derivative 4d binds to DNA much more strongly that the Pro, Ala, and Gly conjugates whereas the Phe analogue showed the lowest DNA binding capacity. These compounds form intercalation complexes with DNA, as judged from electric linear dichroism and topoisomerase I-based DNA unwinding experiments. Preferential binding of 4d to defined sequences such as 5'-CTAAAGG and 5'-ATGC was evidenced by DNase I footprinting. This Lys conjugate was found to be over 20 times more cytotoxic to CEM human leukemia cells than the other conjugates, with an IC50 in the submicromolar range. A high antiproliferative activity, likely attributable to the enhanced DNA binding capacity, is maintained despite the incapacity of the compound to stabilize topoisomerase-DNA covalent complexes. The cell cycle effects of 4d consisted in an S phase accumulation of cells coupled with a pro-apoptotic action (appearance of hypodiploid sub-G1 cells) which were confirmed by measuring the inhibition of BrdU incorporation into DNA and labeling of phosphatidylserine residues with annexin V-FITC by means of flow cytometry. Altogether, the work provides interesting structure-activity relationships in the oxoazabenzo[de]anthracene-amino acid conjugate series and identifies the lysine derivative 4d as a promising candidate for further in vivo evaluation and drug design.
...
PMID:Oxoazabenzo[de]anthracenes conjugated to amino acids: synthesis and evaluation as DNA-binding antitumor agents. 1602 36

DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.
...
PMID:The "GyrA-box" is required for the ability of DNA gyrase to wrap DNA and catalyze the supercoiling reaction. 1633 90

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.
...
PMID:Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix. 1643 17

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase-DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.
...
PMID:Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I. 1658 4

Doxorubicin executes topoisomerase II mediated apoptosis, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores (PTP). To further define the effects of doxorubicin on cell metabolism, we measured cellular respiration, cellular ATP, DNA fragmentation, and cytochrome c leakage in Jurkat (supersensitive), human leukemia-60 (HL-60, sensitive), and HL-60/MX2 (resistant) cells following exposure to 1.0 microM doxorubicin for 30 min. The measurements were made after 24 h of exposure to the drug. In Jurkat and HL-60 cells, doxorubicin treatment increased cellular mitochondrial oxygen consumption and ATP content by 2-3-fold. The increment in oxygen consumption was blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk) and by the PTP inhibitor cyclosporin A. In HL-60/MX2 cells, which are resistant because of a reduced topoisomerase II activity, doxorubicin treatment was without effect on either respiration or ATP content, suggesting that topoisomerase II was essential for induction of apoptosis and stimulation of respiration and ATP content. The conclusion that both of the latter processes were products of oxidations in the mitochondrial respiratory chain was supported by the further observation that rotenone and sodium cyanide inhibited oxygen consumption and substantially lowered ATP content in the treated and untreated cells. Thus, oxidative phosphorylation is enhanced in cells briefly incubated with doxorubicin for as long as 24 h post drug exposure despite apoptosis-associated mitochondrial insults caused by the drug.
...
PMID:Enhanced cellular respiration in cells exposed to doxorubicin. 1674 63

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)]. In solutions containing glucose and cells, [O(2)] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2-4 h of incubation with 20 microM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation.
...
PMID:Inhibition of cellular respiration by doxorubicin. 1691 44


<< Previous 1 2 3 4 5 6 7 8 9 Next >>

---