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)

Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of close-circular DNA molecules differing in their number of superhelical turns (tau) around a mean value of tau in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around tau of DNA molecules of different samples overlappped, the difference in the mean number of superhelical turns from sample to sample, delta tau, could be determined and used to measure the mean number (tau) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian-like distribution of molecules differing by integral numbers around a mean value of tau = -24 +/- 2 at 37 degree C [in 0.2 M NaC1, 10 mM Tris-HC1 (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in tau is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phospholiester bond is closed in vivo. When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsC1-propidium diiodide gradient, the number of delta tau = 24 +/- 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28 degree upon intercalation of one molecule of EtdBr.
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PMID:Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. 17 79

Renaturation of two complementary single-stranded circles should be limited by topological constraints against the rewinding of the DNA helix. If a mixture of complementary single-stranded rings is annealed and then treated with the DNA untwisting enzyme, the DNA circles completely renature as judged by (i) the presence of interlocked rings that sediment at 53 S in alkali, (ii) the buoyant density of the renatured DNA in CsCl gradients containing ethidium bromide, and (iii) the resistance of the product to the single-strand-specific S1 nuclease. Therefore, the DNA untwisting enzyme is able to provide a transient single-strand break that is sufficient to allow the two strands to completely rewind. The possibility that the untwisting enzyme might facilitate the initiation of the process of genetic recombination is discussed.
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PMID:Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme. 20 51

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.
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PMID:Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex. 20 12

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.
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PMID:Mitochondria contain a distinct DNA topoisomerase. 22 16

A DNA-relaxing enzyme capable of concerted nicking and closing of DNA backbone bonds has been purified from Haemophilus gallinarum by two chromatographic steps and gel filtration. The enzyme efficiently catalyzes the removal of superhelical turns from a negatively twisted DNA and requires Mg2+ for this activity. Slight removal of superhelical turns from a positively twisted DNA generated by binding of ethidium bromide is found, but only at high enzyme concentrations. The DNA-relaxing activity is inhibited markedly with heat-denatured DNA, whereas native DNA and RNA have almost no affect on this activity.
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PMID:Purification and characterization of DNA-relaxing enzyme from Haemophilus gallinarium. 22 61

DNase I-hypersensitivity of rat spermatogenic cells was analyzed 1) to establish overall patterns of hypersensitivity in individual cell types, 2) to correlate these patterns with known changes in chromatin organization and function, and 3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from "checkerboard" in steps 7-8 round spermatid nuclei to "lamellar" in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids. DHR were localized throughout the nuclei or in a graded manner--increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicators the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.
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PMID:Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. 131 43

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts. 133 77

Patients with the autosomal recessive disorder Fanconi anemia (FA) present with progressive pancytopenia, skeletal abnormalities and a predisposition to leukemia. In addition to elevated rates of spontaneous chromosome aberrations occurring in cultured fibroblasts and lymphoblastoid cell lines, an increased susceptibility to DNA cross-linking agents and oxygen has been found. To explain this hypersensitivity to clastogenic agents a defective function of DNA topoisomerase I or II could be invoked, a suggestion which is supported by the co-localization of the DNA topoisomerase I gene and a putative FA gene to chromosome 20q. In order to investigate the function of DNA topoisomerases in FA, the sensitivity of lymphoid B-cell lines derived from FA patients and control cell lines to inhibitors of DNA topoisomerases I and II was compared using continuous bromodeoxyuridine labeling and bivariate Hoechst/ethidium bromide flow cytometry. Both agents inhibited cell proliferation mainly by arresting cells in the G2 phase of the cell cycle. However, no difference was found in sensitivity towards both DNA topoisomerase inhibitors between control and FA cell lines.
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PMID:Cell cycle effects of the DNA topoisomerase inhibitors camptothecin and m-AMSA in lymphoblastoid cell lines from patients with Fanconi anemia. 138 35

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.
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PMID:Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes. 169 76

The specificities of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase from brain and liver cells of the chick embryo and of DNase I were demonstrated in vitro by their response to substrate DNA pretreated with monofunctional alkylating agents of different O6-guanine alkylating ability and some antineoplastic agents. Treatment of DNA with ethidium bromide, Hoechst 33258, doxorubicin, Fe2+/bleomycin, and suramin resulted in a dose-dependent diminution of alkyltransferase activity (DE50 approximately 5 micrograms/ml, 15 micrograms/ml, 5 micrograms/ml, 5 micrograms/ml, 100 micrograms/ml, respectively). Apart from bleomycin, comparable results were obtained with DNase I. Thermal denaturation of the substrate DNA reduced both alkyltransferase and DNase I activity. No effect was seen with X-irradiation. Cisplatin decreased only DNase I activity. Some topoisomerase II and/or gyrase inhibitors remained without significant effects on the alkyltransferase reaction whereas DNA catabolism by DNase I was diminished in a dose-dependent manner (DE50 between 6.5 and 19 micrograms/ml).
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PMID:Inhibition of O6-alkylguanine-DNA alkyltransferase and DNase I activities in vitro by some alkylating substances and antineoplastic agents. 172 Jul 84


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