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)

Incorporation of [3H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable. The Triton X-100-extracted nuclei incorporated label into their DNA in nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared. When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [3H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg2+ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei. It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.
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PMID:DNA synthesis in membrane-denuded nuclei and nuclear fractions from host liver and Morris hepatomas. 16 67

A serum protein present in normal rat serum and absent from the serum of hepatoma-bearing animals at advanced stages has a stimulatory effect on 3H-thymidine incorporation into hepatoma cells in suspension. Liver cells maintained in a similar suspension are not affected by the factor. The stimulation appears to be at the level of chromatin or DNA. Isolated membrane-denuded nuclei from Morris hepatoma 7777 incorporate more 3H-TTP when the factor is present in the incubation mixture. Nuclei from host liver are not stimulated. The factor also stimulates incorporation of 3H-TTP in a system using calf thymus DNA as primer and an extracted DNA polymerase. In this system incorporation is stimulated with DNA polymerase from both tissues, host liver and hepatoma 7777. It is concluded that the factor does not act on the DNA polymerase but on chromatin or DNA.
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PMID:Stimulatory effect of a serum factor on DNA synthesis in isolated hepatoma nuclei. 17 43

A new form of high molecular weight DNA polymerase [EC 2.7.7.7] (polymerase N) was isolated from the nuclei of rat ascites hepatoma cells. Polymerase C, which was isolated previously from whole cell extract, was also isolated from the nuclei (Tsuruo, T. and Ukita, T. (1974) Biochim. Biophys. Acta 353, 146-159). Polymerase N was not found in the cytoplasmic fraction of the cell, while polymerase C existed in both the nucleus and cytoplasm. The molecular weight of polymerase N (8.7 S) was larger than that of polymerase C (7.4 S). On freezing and thawing, polymerase N was converted to polymerase C. In the nucleus the amount of polymerase N was larger than that of polymerase C. These data suggest that polymerase N, which was specifically present in the nucleus, was a complex form of polymerase C. In in vitro assay, polymerase N showed properties similar to those of polymerase C. Oligoribonucleotide was an effective initiator for the polymerization reaction by polymerase N. The DNA synthesis on single stranded fd phage DNA was greatly stimulated by the concomitant synthesis of RNA.
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PMID:A new form of high molecular weight DNA polymerase in the nuclei of rat ascites hepatoma cells. 17 84

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.
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PMID:Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase. 18 3

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.
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PMID:Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro. 18 51

The Novikoff hepatoma DNA polymerase-beta sediments as a 7.3S form in crude extracts but during purification sediments as a 4.1S form (after diethylaminoethyl-Sephadex chromatography) or as a 3.3S form (after DNA-cellulose chromatography). If 0.25 M ammonium sulfate or 0.5 M NaCl is included in the sucrose gradients, the 7.3S form sediments at 3.3 S; after removal of the salt, it sediments again at 7.3 S, indicating the reversibility of the aggregation phenomenon. By careful adjustment of ionic strength in the gradient, four distinct and reproducible forms of the enzyme sedimenting at 7.3, 5.8, 4.1, and 3.3 S can be generated. The isoelectric point of the DNA polymerase also changes during purification; the 7.3S form has a pI of 7.5, while the 4.1S form isoelectrically focuses at a pH of 8.5. During DNA-cellulose chromatography, the Novikoff beta-polymerase is separated from a stimulatory factor designated as Novikoff factor IV. Factor IV is a protein as shown by its sensitivity to protease and resistance to nucleases. It is responsible for converting the 3.3S enzyme to the 4.1S form since the 3.3S homogeneous DNA polymerase-beta sediments at 4.1 S in the presence of factor IV. Factor IV confers stability to the polymerase in low ionic strength buffers as well as stability to heat denaturation. Factor IV has the ability to increase the activity of the 3.3S homogeneous polymerase by about fourfold.
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PMID:Novikoff hepatoma deoxyribonucleic acid polymerase. Identification of a stimulatory protein bound to the beta-polymerase. 19 Oct 73

Single-strand breaks induced in DNA of ascitic hepatoma cells by gamma-rays and N-methyl-N-nitrosourea (MNU), resp., may be effectively repaired. Double-strand breaks of DNA from MNU-treated hepatoma cells are also effectively repairable in vivo. Only a small part of double-strand breaks induced by gamma-rays in DNA of these cells is repaired in the postradiation period. The combined action of gamma-rays and MNU on the hepatoma cells causes a complete inhibition of repair of DNA and its further degradation. Under these conditions, inhibition of the repair of DNA synthesis and repression of DNA polymerase I activity is observed.
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PMID:[Degradation and repair of rat hepatoma cell DNA following exposure to gamma rays and methylnitrosourea]. 20 65

A high molecular weight DNA polymerase has been purified from the cytosol of a fast growing hepatoma: LF hepatoma. This enzyme sediments at 11.3 S under polymerization reaction conditions (6 mM KCl) and at 8.3 S in higher salt concentrations (200 mM KCl). In either case, no activity is seen in the 3 to 4 S region where low molecular weight DNA polymerase is found. The purified enzyme has a neutral pH optimum and requires a divalent cation, all four deoxyribonucleoside triphosphates and an initiated DNA template for maximal activity. The synthetic template specificity of LF DNA polymerase has been studied. Although this enzyme cannot copy a polyribonucleotide template, the ribostrand of a synthetic hybrid can be used with low efficiency as an initiator for the synthesis of the complementary deoxyribonucleotide strand. The activity of the purified enzyme is strongly inhibited by thiol-blocking agents. The general properties of LF DNA polymerase are similar to those of high molecular weight mammalian DNA polymerases. In our experimental conditions, the error frequency of this tumoral DNA polymerase was no greater than that made by the purified high molecular weight DNA polymerase of regenerating rat liver.
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PMID:High molecular weight deoxyribonucleic acid polymerase of LF hepatoma. Purification and properties. 24 19

Thymidine kinase, dTMP kinase, and DNA polymerase activities were determined in cell lines of AH hepatoma, L1210 leukemia, and Yoshida sarcoma that were sensitive and resistant to 5-fluorouracil (5-FU). It was found that the levels of these enzymes in tumor cells were not consistently related to the property of sensitivity to 5-FU. A marked difference was observed between sensitive and resistant cell lines of L1210 leukemia and Yoshida sarcoma in the uptake of labeled 5-FU into the acid-soluble, nucleotide, and RNA fractions, the rate of incorporation of 5-FU into these fractions being 3 to 5 times greater in sensitive tumor cells than in resistant tumor cells. The radioactivities in the acid-soluble fractions of AH44 (sensitive) and AH109A (resistant) were similar after incubation of these cells with labeled 5-FU in vitro. However, a smaller volume of ascites and lower cell number were observed in AH44 (sensitive)-bearing rats than in AH109A (resistant)-bearing rats. These in vivo results indicate that the 5-FU injected intraperitoneally was diluted by ascites more in AH109A (resistant)-bearing rats than in AH44 (sensitive)-bearing rats.
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PMID:Metabolism of 5-fluorouracil in sensitive and resistant tumor cells. 55 50

Chromatin prepared from S phase hepatoma tissue culture (HTC) cell incorporates in vitro about 11-14 pmoles [3H]dTMP into DNA in 30 min. Single-stranded DNA added to this chromatin stimulates DNA synthesis more than 40-fold whereas activated DNA enhances it about 60-fold. By contrast, stimulation of DNA synthesis by activated DNA in a crude nuclear extract exceeds the stimulation exerted by denatured DNA by a factor of 7. Stimulation of DNA synthesis by denatured DNA is not due to stabilization of either the chromatin or the product of the endogenous reaction. On the other hand, we find that poly(dC) and poly (dT) enhance DNA synthesis by serving as templates which are copied by chromatin in a true complementary fashion. It seems therefore, that eukaryotic cell chromatin is able to copy single-stranded DNA at a high efficiency. Chromatin of G1 arrested cell copies exogenous templates at a considerably reduced rate. The enzyme responsible for the copying of denatured DNA is tentatively identified as DNA polymerase alpha on the basis of its sensitivity to sulfhydril group blocking, its requirements for ions and failure to copy the ribo strand of oligo(dT) poly(A).
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PMID:Highly efficient copying of single-stranded DNA by eukaryotic cell chromatin. 67 65


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