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)

Hydroxyurea is a potent inhibitor of the enzyme ribonucleotide reductase. Due to its effects on cellular deoxyribonucleotide pools, hydroxyurea can modulate the activity of several pyrimidine and purine antimetabolites. As an inhibitor of DNA repair, it can potentially interact with DNA-damaging agents such as alkylating agents or inhibitors of topoisomerase II. Both cytokinetic and biochemical interactions occur between hydroxyurea and cytarabine (ara-C), which account for their synergistic cytotoxicity. Inhibition of ribonucleotide reductase by hydroxyurea depletes cellular deoxycytidine triphosphate pools, thereby enhancing ara-C uptake and phosphorylation to ara-C triphosphate. In a phase II clinical trial, the combination of hydroxyurea and ara-C produced a 43% response rate in patients with refractory malignant lymphoma. Studies in murine leukemia models have demonstrated therapeutic synergy when hydroxyurea is combined with fluoropyrimidines. High levels of deoxyuridine monophosphate that have been associated with resistance to 5-fluorouracil can be suppressed by hydroxyurea, leading to greater inhibition of thymidylate synthase. Despite the strong biochemical rationale for the use of hydroxyurea and 5-fluorouracil in combination, few clinical trials have been conducted thus far. Antimetabolites and topoisomerase II inhibitors have also been shown to be synergistic in vitro. Hydroxyurea has been shown to enhance the formation of DNA strand breaks produced by amsacrine and to produce synergistic cytotoxicity with etoposide. A phase I clinical trial of these drugs has demonstrated bone marrow suppression to be the major toxicity of the combination. In summary, hydroxyurea has been shown to undergo cytokinetic and biochemical interactions with a number of established antitumor agents. Clinical trials of hydroxyurea in combination with these agents have identified doses and schedules of administration that produce acceptable levels of clinical toxicity and appear feasible for further testing.
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PMID:Laboratory and clinical studies of biochemical modulation by hydroxyurea. 164 59

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.
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PMID:Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells. 165 49

Bacteriophage T4 ribonucleoside diphosphate reductase consists of alpha 2 and beta 2 subunits encoded by genes nrdA and nrdB, respectively, and plays a central role in the T4-induced deoxyribonucleotide synthetase complex. The accompanying paper describes the decreased rate of synthesis of deoxyribonucleotides after infection by the T4 mutant, nrdB93, and the suppression of this defect by a second mutation in gene 39, coding for one of the three protein chains of T4 DNA topoisomerase. In this study we examined these effects at the protein level. On infection by nrdB93 not only was the beta 93 protein chain altered, as shown by its migration relative to the wild type protein in electrophoretic gels and by its temperature sensitivity, but the infected cells showed very low levels of the protein. However, on infection with the double mutant of nrdB93 and 39-01 (gene 39) the concentration of beta 93 chain returned to the values of beta protein found with wild type phage. A double mutant bearing nrdB93 and an amber mutation of gene 39 also suppressed the nrdB93 defect. By contrast, a temperature-sensitive mutant of gene 39, A41, did not show suppression at either 30 or 41 degrees C. Amber mutations in the two other genes coding for T4 DNA topoisomerase, 52 and 60, did not suppress the defect. We propose that the deficiency in the quantity of beta 93 chain and the suppression of this defect occur at the transcriptional or translational expression of the nrdB93 gene and that a specific domain of the gene 39 protein, not acting in the capacity of T4 DNA topoisomerase, inhibits the expression.
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PMID:Effect of bacteriophage T4 DNA topoisomerase gene 39 on level of beta chain of ribonucleoside diphosphate reductase in a T4 nrdB mutant. 283 67

Detergent-soluble DNA is the fraction (2-4%) of DNA that is released into the supernate upon mild detergent lysis. It is nonmitochondrial in origin. It labels efficiently with deoxy[3H]ribonucleosides and the labeling is prevented by inhibitors of polymerase alpha and ribonucleotide reductase. In previous publications we have characterized detergent-soluble DNA from splenocytes of immunologically activated mice. In this publication we show that incorporation of [3H]thymidine into detergent-soluble DNA is prevented by pretreatment with novobiocin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and teniposide (VM26), three inhibitors of type II topoisomerases. Camptothecin, an inhibitor of type I topoisomerases, also reduces incorporation of [3H]thymidine but only to 50% of control levels. In addition to affecting incorporation of [3H]thymidine, preincubation with the topoisomerase II inhibitors m-AMSA and VM26 alters the amount of DNA recovered in the detergent-soluble fraction. At low concentrations of m-AMSA the amount of detergent-soluble DNA increases somewhat, whereas at higher drug concentrations a marked decrease is observed. Treatment with VM26 results in diminished amounts of DNA being released into the detergent-soluble fraction as well. However, maximal inhibition of detergent-soluble DNA release by VM26 requires the presence of camptothecin. Therefore, we suggest that topoisomerases play an important role in making a small part of lymphocyte chromatin detergent labile. Furthermore, these results are consistent with recent studies demonstrating a role for topoisomerases in yeast replication. Thus, the newly synthesized portion of detergent-soluble DNA may arise as DNA replication intermediates not yet stabilized into mature chromatin.
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PMID:Role for topoisomerases in the release of DNA into the detergent-soluble fraction of eukaryotic cells. 301 25

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).
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PMID:Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. 629 95

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.
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PMID:The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells. 632 Oct 50

We have previously shown that a fraction from the nuclei of S phase (DNA-synthesizing) Chinese hamster embryo fibroblasts (CHEF/18 cells) can be obtained that has a number of the enzyme activities required for DNA biosynthesis, and can catalyse the incorporation of labelled precursors into DNA (refs 1-4, also see ref. 8). This fraction, which we have termed the 'replitase', contains spherical particles of diameter approximately 25 nm, apparently multienzyme complexes for de novo DNA biosynthesis. Here we present evidence for the functional association of one of the enzyme activities, thymidylate synthase, with several of the other enzyme activities. Hydroxyurea, novobiocin and aphidicolin, inhibitors of ribonucleotide reductase, topoisomerase and DNA polymerase alpha, respectively, all inhibit thymidylate synthase in intact S phase CHEF/18 cells, but not in their soluble extracts. We suggest that these results reflect allosteric interactions between the subunits of a multienzyme DNA-synthesizing complex, which can be modulated by the specific inhibitors of individual enzyme activities in intact cells.
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PMID:Inhibitor evidence for allosteric interaction in the replitase multienzyme complex. 640 86

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
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PMID:Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides. 675 52

Bacteriophage T4 ribonucleoside diphosphate reductase is composed of two proteins, alpha 2 and beta 2, encoded by the nrdA and nrdB genes, respectively. The expression of nrdB is the limiting factor for the assembly of the enzyme. A recently described mutation, nrdB93, may give new insight into the regulation of synthesis of the beta subunit encoded by nrdB. Infection by T4 nrdB93 produced only low concentrations of the beta 2(93) protein. However, a site-specific mutation of phage T4 gene 39, encoding one of the subunits of T4 DNA topoisomerase, phenotypically suppressed the defect. The present work sought to characterize the nature of this defect. The mutation in nrdB93 was a single-base transition (G-->A) resulting in a Gly253-->Asp change. In vivo and in vitro studies provided no evidence of degradation of the beta 2(93) protein. Furthermore, the decrease in beta 2(93) formation was not caused by a delayed onset of transcription, neither by a decreased rate of mRNA formation from the nrdB promoter, nor by a defective intron splicing of the nrdB gene or in the transcription of the terminal segments of the message. These findings are consistent with the concept that the nrdB93 lesion produces a defect at the level of translation.
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PMID:Bacteriophage T4 ribonucleoside diphosphate reductase: on the defect causing decreased formation of the beta 93(2) subunit encoded by the nrdB93 mutant gene. 818 57

Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.
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PMID:Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes. 880 83


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