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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.
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PMID:Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells. 231 56

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

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.
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PMID:The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene. 253 61

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

In recent years, evidence has accumulated that suggests that mammalian topoisomerase may play a role in the formation of spontaneous or chemically induced sister chromatid exchange (SCE). In microbial systems, nalidixic acid is known to disrupt the function of a topoisomerase-like enzyme, DNA gyrase. To explore the possible relationship to topoisomerase function and SCE formation in mammalian cells, an analog of nalidixic acid with potent topoisomerase II inhibitory activity was selected for examination in a variety of genetic toxicology assays. This analog, CP-67,015, proved to be a positive direct-acting mutagen in the L5178Y/TK+/-, CHO/HGPRT, and V79/HGPRT systems. However, no gene mutational activity was observed using the Ames test in direct plate, mouse and rat metabolic activation, and mouse urine tests. In vitro cytogenetic studies showed strong clastogenic activity in human lymphocytes and in CHO cells. Compound-induced chromosome damage was also observed in vivo in mouse bone marrow cells. Surprisingly, SCE studies in vitro in human lymphocytes or CHO cells showed only slight increases, even at levels producing severe chromosome breakage. Mouse bone marrow showed no significant elevation of SCE following parenteral treatment with CP-67,015. These results, taken together, demonstrate that CP-67,015 is a direct-acting mutagen in mammalian cells with both gene and chromosomal level effects. The relative ineffectiveness in producing SCEs suggests that CP-67,015 may interfere with a DNA replicative/repair process, perhaps by alteration of one or more DNA polymerase activities. This suggestion is based in part on the known effect of the analog nalidixic acid on DNA gyrase in microbial cells and on topoisomerase in mammalian cells. The profile of genetic activity of CP-67,015, coupled with its inhibitory effect on topoisomerase function, gives rise to a model for SCE formation that is based on anomalies of topoisomerase activity during DNA synthesis.
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PMID:Genetic profile of a nalidixic acid analog: a model for the mechanism of sister chromatid exchange induction. 253 98

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.
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PMID:Studies on DNA polymerases and topoisomerases in archaebacteria. 254 77

Fredericamycin is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro. Because of its unique structure and the absence of a clearly defined mechanism of action, we examined the effects of this compound on L1210 cells in culture as well as on several enzymes that bind to DNA. Fredericamycin exhibits an IC50 of 4.4 microM toward L1210 cells, and its cytotoxicity is a function of the time of exposure as well as drug dose. No DNA breakage was observed in L1210 cells or isolated nuclei following exposure to highly lethal concentrations of fredericamycin. As a first step toward understanding its mechanism of action, we examined the effect of fredericamycin on several enzymes involved in DNA metabolism. The catalytic activity of both DNA topoisomerases I and II were totally inhibited by fredericamycin concentrations of 4.4 and 7.4 microM, respectively. Fredericamycin blocked etoposide-stimulated DNA cleavage by topoisomerase II both in vitro and in isolated nuclei. In addition, the drug inhibits DNA polymerase a in vitro, exhibiting an IC50 of 93 microM. These diverse actions of fredericamycin do not enable us to draw conclusions regarding its mechanism of antitumor effect but clearly identify it as a compound of pharmacologic interest.
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PMID:Inhibition of topoisomerases by fredericamycin A. 254 7


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