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 presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

The RNA-directed DNA polymerase of Rous sarcoma virus requires a 4S RNA molecule as primer for the initiation of DNA synthesis on the viral 70S RNA genome. We have now functionally identified primer activity in uninfected cells on the basis of the capacity of cellular 4S RNA to actively participate in the initiation of DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus in vitro. This was accomplished by reconstitution experiments in which 4S RNA from uninfected avian cells was tested for its ability to restore template activity to the viral RNA genome from which all primer had been removed. Similar reconstitution experiments were employed to demonstrate a primer activity in the 4S RNA population of duck, mouse, and human cells. Primer activity appears to be absent in lower eukaryotic or prokaryotic cells. Unambiguous identification of the Rous sarcoma virus primer molecule in uninfected cells was accomplished by directly purifying a 4S RNA molecule from the bulk of host cell transfer RNA and establishing structural similarities between this cellular 4S RNA species and the Rous sarcoma virus primer by two-dimensional paper electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the RNA species. We conclude that the Rous sarcoma virus DNA polymerase can utilize a host cell molecule as primer for the initiation of RNA-directed DNA synthesis in vitro.
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PMID:RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells. 4 51

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with dexamethasone, a high-speed pellet of the supernatant fluid and disrupted cells was prepared and analyzed on a sucrose gradient for enzymatic activity characteristic of RNA-directed DNA polymerase (reverse transcriptase). Peaks of endogenous DNA polymerase activity showing ribonuclease sensitivity and/or stimulation with the synthetic template poly(rC)-(dG)12-18 were demonstrated in both patients at densities of 1.15 to 1.19 and 1.21 to 1.24 g/ml. Subsequently, diagnosis 2 and 4 months after initial evaluation revealed acute myelogenous leukemia and malignant histiocytosis, respectively. Prior studies have suggested a possible etiological significance of such particles in human leukemia. The demonstration of similar particles preceding clinically overt disease in these patients supports this hypothesis and offers the possibility of early diagnosis and treatment.
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PMID:Oncornavirus-like particles from cultured bone marrow cells preceding leukemia and malignant histiocytosis. 5 58

Measurement of DNA polymerase in leukaemic guinea-pig plasms reveals the presence of low levels of sedimentable and non-sedimentable enzymic activities. Since the sedimentable DNA polymerase is ribonuclease sensitive, uses poly(C).oligo(dG) as template, and bands in a sucrose density gradient at 1-17 g/ml it is thought to be the GPLV-associated reverse transcriptase. The soluble DNA polymerase is stimulated by ribonuclease and is probably of cellular origin.
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PMID:DNA polymerase activity in plasma from leukaemic guinea-pigs. 5 90

When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
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PMID:Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. 16 2

Extracellular particles, with a density of 1.18-1.22 g/cm3 in sucrose, were detected in the culture medium of a continuous cell line (JIII) derived from a patient with monocytic leukemia. These particles contained RNA, DNA, and a DNA polymerase. They synthesized DNA with endogenous templates and primers and also used exogenous DNA but not poly(rC) oligo(dG) as a template. Pretreatment with Nonidet P-40 stimulated DNA polymerase activity while treatment with ribonuclease partially inhibited the enzyme activity. Fluorescent antibodies made to the particles stained both JIII and Z-597 cells derived from human leukemias but not other types of human or nonhuman cultured cells tested. The particles do not appear to be oncornaviruses but may be a particulate antigen associated with malignant cells of hemopoietic and lymphoid origin.
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PMID:Characterization of extracellular particles released from continuous cell cultures derived from human leukemia. 18 75

Several newly synthesized boron betaine analogs had antitumor activity in Ehrlich ascites, Walker 256 ascites carcinosarcoma, and Lewis lung screens and marginal activity in the B-16 melanotic melanoma screen. In vivo testing demonstrated that trimethylamine-cyanoborane inhibied Ehrlich ascites cell DNA and protein syntheses as well as gene modulation by chromatin protein phosphorylation and methylation. Trimethylamine-cyanoborane increased cyclic-AMP levels. In vitro testing showed that nuclear DNA polymerase, thymidylate synthetase, S-adenosylmethyltransferase, nonhistone chromatin methylation, deoxyribonuclease, ribonuclease, and cathepsin were inhibited by the boron analogs. These compounds did not demonstrate high antitumor activity at the doses employed, but blockage of methyl transfer from S-adenosylmethionine was established as a feasible method for controlling cell proliferation.
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PMID:Boron betaine analogs: antitumor activity and effects on Ehrlich ascites tumor cell metabolism. 22 87

Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.
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PMID:Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis. 37 16

A ribonuclease-sensitive DNA polymerase, which uses an endogenous template, is detectable in the 39,000 g supernatant of a rat thymus homogenate, and appears as a single peak of activity in the void volume after Sephadex G 150 or G 200 gel filtration chromatography. Native and "activated" DNA-dependent DNA polymerase activities coincide with the endogenous-templated polymerase activity. Treatment of the thymus extract with ribonuclease(s) prior to gel filtration chromatography yields two other peaks of activity in addition to the void volume peak. The appearance of the two lower molecular weight peaks of activity is accompanied by a concomitant decrease in the endogenous-templated activity. The effect of ribonuclease is specific and cannot be reproduced by a similar deoxyribonuclease treatment.
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PMID:DNA polymerase activity associated with endogenous template: release by ribonuclease treatment. 80 37

An endogenous DNA-synthesizing complex sensitive to ribonuclease has been found in purified preparations of swollen human sperm heads. Incorporation of [3H]dTTP into acid-precipitable material occurred in the presence of actinomycin D and required addition of dGTP, dCTP, dATP, plus Mg++. Polymerization was sensitive to pretreatment of the complex with pancreatic RNase A or Triton X-100. Exogenous activity was elicited by the synthetic template (dT)12--18-(rA)n but not by (dT)12--18-(dA)n or (dT)10. The complex sedimented from a 10,000 X g supernatant by centrifugation at 165,000 X g for 60 min and banded in sucrose at a density of 1.21--1.25 g/cm3. Endogenous RNase-sensitive DNA polymerase activity from cell-free seminal fluid was also detected in a fraction in sucrose at a density of 1.22 g/cm3. This activity was labile to freezing and stimulated by 0.04% Triton X-100, and thus differed from that of sperm heads.
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PMID:Ribonuclease-sensitive DNA-synthesizing complex in human sperm heads and seminal fluid. 105 11


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