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)

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.
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PMID:Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene. 138 64

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

Enhanced DNA repair has been identified as a major mechanism of resistance to the anticancer drug cisplatin in murine leukemia L1210 cells. Studies of other cells have implicated the elevation of a variety of RNA transcripts in cisplatin resistance. This study investigated potential changes in transcription of these genes as well as genes involved in DNA repair. No elevation in any of the following transcripts was observed: thymidylate synthase, dihydrofolate reductase, DNA polymerase alpha, DNA polymerase beta, topoisomerase II, Ha-ras, beta-tubulin, metallothionein and the DNA repair genes ERCC1 and ERCC2. Thymidine kinase was increased no more than 2-fold. None of these RNA were induced by incubation with cisplatin. High levels of cisplatin produced selective decreases in certain RNA. These results demonstrate that the previous observations of elevated RNA can not be universally applied to all cisplatin-resistant cells.
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PMID:Analysis of various mRNA potentially involved in cisplatin resistance of murine leukemia L1210 cells. 197 66

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.
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PMID:Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta). 217 36

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.
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PMID:DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system. 221 24

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.
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PMID:Purification and characterization of primer recognition proteins from HeLa cells. 236 57

Cultured human epidermal keratinocytes were used as a model system for testing compounds with potential therapeutic effect against hyperproliferative skin disorders. We have investigated whether each test compound caused direct damage to the DNA or inhibited DNA repair and/or seminconservative replication of DNA, as well as its effect on the overall rate of protein synthesis and on expression of specific keratin genes. The following compounds were studied: (a) inhibitors of DNA polymerase alpha [aphidicolin and its derivative aphidicolin glycine], (b) inhibitors of topoisomerases [novobiocin, nalidixic acid, teniposide, etoposide, and 4'-(9-acridylamine) methanesulfon-m-anisidide], (c) modifiers of chromatin structure [sodium butyrate, 3-aminobenzamide, and nicotinamide], (d) inhibitors of calmodulin activation and protein kinase C [chlorpromazine and trifluoperazine]; and (e) drugs used in clinical dermatology [anthralin, fluocinolone acetonide, ketoconazole, and hydroxyurea]. The compounds were tested at concentrations at which they were known from the literature to be effective in their respective actions. Among the groups of compounds studied, the topoisomerase inhibitors were particularly interesting since they caused no detectable damage to DNA but exhibited maximal inhibitory effect on replication combined with minimal inhibition of DNA repair. In addition most of the topoisomerase inhibitors, particularly novobiocin, changed the pattern of gene expression by inhibiting the synthesis of certain keratins and inducing a Mr 67,000 protein in the prekeratin fraction. These properties combined with minimal systemic side effects may encourage the clinical exploration of some topoisomerase inhibitors for antiproliferative therapy of skin disorders.
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PMID:Comparative effects of growth inhibitors on DNA replication, DNA repair, and protein synthesis in human epidermal keratinocytes. 242 88

Nalidixic acid, a DNA topoisomerase inhibitor, has been reported to inhibit DNA repair in some mammalian systems. To investigate the effect of nalidixic acid on DNA repair in cultured rat hepatocytes, DNA damage was induced by ultraviolet light or N-methyl-N-nitro-N'-nitrosoguanidine. The presence of aphidicolin, a DNA polymerase alpha inhibitor resulted in a decrease in DNA repair. Nalidixic acid had no inhibitory effect. Neither aphidicolin nor nalidixic acid induced DNA repair. These results indicate that nalidixic acid does not damage DNA or inhibit DNA repair processes in hepatocytes.
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PMID:Effect of nalidixic acid on DNA repair in rat hepatocytes. 250 47

Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.
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PMID:Identification of cellular proteins required for simian virus 40 DNA replication. 253 23

Our earlier studies have shown that gossypol [1,1',6,6',7,7'-hexahydroxy-5,5-diisopropyl - 3,3'-dimethyl - (2,2'- binaphthalene)-8,8'-dicarboxyaldehyde], a male contraceptive, inhibits DNA synthesis by decreasing the activities of DNA polymerase alpha and beta, resulting in the arrest of cells in mid-S phase [L.J. Rosenberg, R.C. Adlakha, D.M. Desai, and P.N. Rao, Biochim. Biophys. Acta, 866: 258-267, 1986]. Now we have examined the effects of gossypol on another enzyme of importance to cellular functions, topoisomerase II (topo II). We have determined the consequences of gossypol treatment on 4'-(9-acridinylamino)methane-sulfon-m anisidide (m-AMSA)-induced topoisomerase II-mediated, protein-associated DNA cleavage using the alkaline elution technique. In HeLa cells pretreated with gossypol (3.4-17.5 microM) for 8-16 h we observed a dose- and time-dependent decrease (50-75%) in DNA cleavage compared to that quantified in cells treated with m-AMSA alone. Gossypol by itself did not induce more than 25 rad-equivalents of DNA single-strand breaks even at the highest dose tested (26 microM). [14C]m-AMSA uptake was identical in treated and untreated cells. Pretreatment of cells with another inhibitor of DNA synthesis, thymidine, which blocks cells at G1/S boundary increased the m-AMSA-induced DNA cleavage by 25%, suggesting that the effect of gossypol might be due to the arrest of cells in mid-S phase. In contrast to gossypol's effects on m-AMSA-induced DNA cleavage, m-AMSA-induced cytotoxicity was actually increased in gossypol pretreated cells. Gossypol blocked topo II strand passing activity (decatenation of kinetoplast DNA) of cellular extracts from HeLa cells. The inhibition of this activity by gossypol was synergistic with the inhibition produced by m-AMSA or etoposide. These data suggest that gossypol can both inhibit topo II catalytic activity and interfere with the stabilization of topo II-DNA complex formation by m-AMSA. These data indicate that the magnitude of m-AMSA-induced DNA cleavage may not necessarily parallel the magnitude of m-AMSA-induced cytotoxicity. The cytotoxicity data may rather be explained by an action of gossypol and m-AMSA to block topo II catalytic activity at a point in the enzyme's strand passing cycle prior to cleavage complex formation that might be particularly toxic to cells in S phase. Gossypol should therefore be useful in improving our understanding of the cellular role of topo II and the consequences of interference with topo II activity by active antineoplastic agents.
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PMID:Modulation of 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced, topoisomerase II-mediated DNA cleavage by gossypol. 253 51


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