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 uracil-DNA glycosylase inhibitor gene (ugi) of the Bacillus subtilis bacteriophage PBS2 has been subcloned to a 720-base pair DNA fragment contained in pZW2-0.7 and its nucleotide sequence determined. Using nucleotide deletion analysis, we have located the cloned ugi gene along with potential regulatory elements. A promoter-like region (-10 and -35 consensus sequences) similar to other B. subtilis genes and the Shine-Dalgarno sequence characteristic of Gram-positive bacteria were both identified upstream from the initiator AUG codon. A 17-nucleotide exact inverted repeat followed by runs of adenine and thymine residues was positioned almost immediately downstream of the ochre codon. The ugi gene product was identified on sodium dodecyl sulfate-polyacrylamide gels using Escherichia coli minicells containing pZW2-0.7 and by recovering uracil-DNA glycosylase inhibitor activity following electrophoresis. The ugi gene codes for an acidic polypeptide of 9,477 molecular weight (84 amino acids) whose electrophoretic mobility was greater than predicted for a protein of this size. The mode of inhibition did not appear to involve a catalytic process nor did it directly involve inhibitor-DNA interaction. Rather, the inhibitor protein was shown to bind physically to the E. coli uracil-DNA glycosylase, forming a 36,000 molecular weight complex. This complex seems to be reversible, since inhibitor activity was recovered after heat treatment of the complex. In addition, we demonstrated that the inhibitor protein is active against uracil-DNA glycosylases isolated from several diverse biological sources but inactive against E. coli deoxyuridine triphosphatase, DNA polymerase I, and DNA polymerase alpha, beta, and gamma.
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PMID:Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. 249 16

The responsiveness and action mechanisms of steroid hormones and epidermal growth factor on human endometrial carcinoma cells are analyzed by using in vitro culture system. 1) The Ishikawa cells, derived from a well differentiated endometrial adenocarcinoma and possess ER and PR, are shown to respond to estrogens by increasing a variety of parameters, viz cell proliferation, PR levels, ALP and DNA polymerase activities. 2) ER and PR of those cells are localized in the nuclei by immunocytochemical staining using the monoclonal antibodies against to ER and PR, confirming the correctness of Gorski and Greene's one step theory involving the action mechanisms of steroid hormones. 3) Progestins reduced the ER level and stimulate E2DH activities and glycogen content, which are completely abolished by anti-progestin (RU486), suggesting that PR of those cells should be functional. 4) These responses to steroid hormones of Ishikawa cells are synergistically enhanced or appeared earlier by addition of EGF. 5) The main metabolite of E2 incubated with Ishikawa cells is E2-3-sulfate instead of E1, indicate that the higher estrogenic status may be persisted in endometrial cancer tissues.
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PMID:[Responsiveness and mechanisms of action of steroid hormones in human endometrial adenocarcinoma cells]. 251 14

A new cytotoxic acridine alkaloid that exhibited antitumor activity in vivo was isolated from a marine Dercitus species sponge collected at a depth of 160 m in the Bahamas. This violet alkaloid, designated dercitin, inhibited the proliferation of cultured murine and human leukemia, lung, and colon tumor cells at nM concentrations (IC50 values of 63-150 nM) and prolonged the life of mice bearing ascitic P388 tumors (%T/C = 170, 5 mg/kg, i.p., QD1-9). Dercitin was also active against i.p. B16 melanoma and modestly inhibited the growth of s.c. Lewis lung carcinoma on the same schedule. DNA blocked the antiproliferative effects of the agent in culture, and incorporation studies indicated that dercitin disrupted DNA and RNA synthesis with less effects on protein synthesis, similar to the effects of known DNA intercalators. After 1-h exposure to 400 nM dercitin, the rates of incorporation of [3H]uridine, [3H]thymidine, and [3H]leucine by cultured P388 cells were inhibited 83, 61, and 23%, respectively. Equilibrium dialysis indicated that dercitin bound calf thymus DNA with an affinity of 3.1 microM and maximal binding of 0.20 mol dercitin/mol base pair. Binding involved intercalation as evidenced by ability to relax supercoiled phi X174 DNA (half maximal concentration for dercitin relaxation was 36 nM). The effects of dercitin on DNA mobility were reversible, and complete relaxation of DNA with topoisomerase I in the presence of dercitin followed by phenol extraction resulted in the appearance of supercoiled DNA. Dercitin, at microM concentrations, had a small effect in the K+-sodium dodecyl sulfate assay using cultured P388 cells, suggesting minimal inhibition of topoisomerase activity. But, dercitin completely inhibited DNA polymerase I/DNase nick translation of DNA at 1 microM. Relaxation of DNA at a given concentration was greater than inhibition of nick translation suggesting that the effects of dercitin on enzyme activity were secondary to changes in DNA conformation. Results indicate that dercitin is a new marine natural product that probably exerts its biological effects through intercalation into nucleic acids.
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PMID:Antitumor activity and nucleic acid binding properties of dercitin, a new acridine alkaloid isolated from a marine Dercitus species sponge. 254 17

Phosphonoacetic acid (PAA) inhibits the replication of human herpesvirus-6 (HHV-6) in mononuclear cells from cord bloods which are susceptible for natural HHV-6 infection in humans. Nuclear extracts of uninfected or HHV-6-infected mononuclear cells were applied to phosphocellulose column chromatography, and DNA polymerase activity was measured with or without the addition of 100 mM ammonium sulfate. The major DNA polymerase activities eluted at 0.47 M KCl were suppressed in both uninfected and HHV-6 infected cells by the addition of 100 mM ammonium sulfate. DNA polymerase activity eluted at 0.47 M KCl was observed only from HHV-6-infected cells; it was enhanced by 100 mM ammonium sulfate and neutralized with immune serum. DNA polymerase activity eluted at 0.73 M KCl was determined to be HHV-6 specific and had the properties of a typical herpesvirus-induced DNA polymerase. PAA inhibited HHV-6-specific DNA polymerase activity.
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PMID:Phosphonoacetic acid inhibits replication of human herpesvirus-6. 256 36

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.
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PMID:HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells. 257 75

DNA polymerase gamma has been purified over 10,000-fold from mitochondria of Xenopus laevis ovaries. We have developed a novel technique which specifically photolabels DNA polymerases. This procedure, the DNA polymerase trap, was used to identify a catalytic subunit of 140,000 Da from X. laevis DNA polymerase gamma. Additional catalytically active polypeptides of 100,000 and 55,000 Da were identified in the highly purified enzyme. These appear to be products of degradation of the 140,000-Da subunit. The DNA polymerase trap, which does not require large amounts of enzyme or renaturation from sodium dodecyl sulfate, is an alternative to the classic "activity gel."
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PMID:DNA polymerase gamma from Xenopus laevis. I. The identification of a high molecular weight catalytic subunit by a novel DNA polymerase photolabeling procedure. 260 77

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCIIOslo) from the plasma of ne of these individuals. HCIIOslo or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G----A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.
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PMID:Heparin cofactor IIOslo. Mutation of Arg-189 to His decreases the affinity for dermatan sulfate. 264 47

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits growth proliferation of human bone marrow progenitor cells in vitro [Antimicrob. Agents Chemother. 31:452-454 (1987)]. The present study evaluates the effect of toxic concentrations of AZT on possible sites of toxicity in human bone marrow cells. Exposure of cells over a 6-hr period to AZT concentrations between 0.5 and 50 microM resulted in a decreased incorporation of tritiated deoxyguanosine into DNA. Unchanged AZT and its phosphorylated metabolites accumulated within cells after exposure to 10 microM [3H]AZT. 3'-Azido-3'-deoxythymidine-5'-monophosphate was the predominant metabolite, reaching a concentration of 49.2 +/- 14.1 pmol/10(6) cells after 48 hr, and a continuous increase was observed in all phosphorylated derivative levels between 2 and 48 hr of incubation. Using a highly sensitive and specific DNA polymerase assay, endogenous deoxyribonucleotide pool size(s) were analyzed for 48 hr after incubation of cells with a pharmacologically relevant concentration of 10 microM AZT. After a 6-hr exposure, 2'-deoxycytidine-5'-triphosphate and 2'-deoxythymidine-5'-triphosphate pools represented approximately 86 and 70% of the control values; levels returned to normal after 24 hr and remained subsequently unchanged. Nucleic acids of human bone marrow cells exposed for 24 hr to 10 microM [3H]AZT were purified and analyzed by cesium sulfate density gradient. No radioactivity was detected in the RNA region, whereas a significant amount was associated with the DNA region. Hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated specific incorporation of AZT into DNA. In additional studies, the amount of AZT incorporated into DNA was correlated with the initial extracellular AZT concentration. In particular, a significant relationship (p less than 0.0001) between the level of AZT incorporated into DNA and the inhibition of clonal growth was observed at concentrations of AZT between 1 and 25 microM (IC50 and IC85 for human bone marrow cells). In summary, these studies demonstrate that AZT is incorporated into DNA of human bone marrow cells and suggest that incorporation of AZT into DNA may be one mechanism responsible for AZT-induced bone marrow toxicity. In contrast, imbalance of deoxyribonucleotide pools by AZT appears unlikely to be associated with inhibition of DNA synthesis and toxicity in human bone marrow cells.
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PMID:Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. 274 33

The DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.
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PMID:Purification and properties of a specific primase-stimulating factor of bovine thymus. 283 71

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
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PMID:Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. 284 66


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