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)

Enzymes of deoxyribonucleotide and DNA biosynthesis, which are little known in plants, were studied in root tips of germinating broad beans (Vicia faba) and in fast-growing cultures of soybean cells (Glycine max). The plant cells contain a ribonucleoside 5'-diphosphate reductase which is detected in vitro only during a limited period of growth, viz. 30--32 h after inhibition of Vicia seeds, and between the second and third day after inoculation of soybean cultures. In both species ribonucleotide reductase activity precedes maximum DNA synthesis. The reductases could be precipitated with ammonium sulfate but were not purified further due to the extremely low enzyme content of the plant extracts. Therefore the reductive pathway of deoxyribotide formation was also established in Vicia root tips by efficient labeling of the plant DNA with a ribonucleoside, [5-3H]cytidine, which reaches a maximum at the same time as the reductase activity measured in vitro. Cycloheximide inhibits this process, indicating the need for de novo enzyme induction. In contrast, DNA polymerase is present in the tissue throughout the entire development and rises only 2-fold in activity during the S phase. The soluble polymerases were partially characterized in both legume species and were found very similar to the DNA polymerase of pea seedlings. Ribonucleotide reductase is more likely a limiting component of DNA formation during the plant cell cycle than DNA polymerase.
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PMID:Deoxyribonucleotide synthesis and DNA polymerase activity in plant cells (Vicia faba and Glycine max). 42 Aug 54

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.
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PMID:Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex. 42 7

Continuous exposure to inhibitory concentrations of methotrexate produces distinct rates of steady-state growth of murine leukemia L1210 and human leukemia CCRF-CEM cells in culture. Addition of thymidine to the medium produces reversal (6 to 40%) of this steady-state growth rate inhibition. This study utilized combinations of methotrexate and thymidine for an evaluation of the accompanying relationship between steady-state growth rate and changes in the ribo- and deoxyribonucleoside triphosphate pools. In L1210 cells exposed to methotrexate alone, the deoxythymidine 5'-phosphate (dTTP) pools decreased, whereas deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and deoxycytidine 5'-triphosphate (dCTP) remained relatively constant up to 70% inhibition of growth rate, with dCTP at a constant 112% of controls. The corresponding ribonucleoside triphosphates decreased only slightly. With the combination of methotrexate and thymidine resulting in up to 40% inhibition of growth rate, there was also a decrease in the dTTP pool while the other deoxyribonucleoside triphosphates remained relatively constant, and the corresponding ribonucleoside triphosphates again decreased only slightly. The dCTP pool was reduced to a constant 42% of control comparable to that produced by thymidine alone. With greater than 40% (with thymidine) or 70% (without thymidine) inhibition of growth rate, all pools decreased, but only dTTP was substantially reduced in proportion to the growth rate inhibition caused by methotrexate. The dTTP pool became depleted in spite of the presence of exogenous thymidine. Evaluation of CCRF-CEM cells indicated that inhibition of growth rate and nucleotide pool perturbations by methotrexate were similar to those observed in L1210 cells. However, in the presence of thymidine, inhibition of growth rate appeared related to decreased pools of dCTP, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate, rather than dTTP as was observed for L1210 cells. Hence, mammalian cells were capable of responding in a differential fashion to pharmacological perturbations, and this capacity may play a role in determining therapeutic selectivity. Since the ribonucleoside triphosphate decreases were slight and relatively uniform during methotrexate-induced perturbations, the deoxyribonucleoside triphosphate pools appear to be more directly related to inhibition of growth rate. The results are consistent with the concept that slight imbalances in the deoxyribonucleoside triphosphate pools dramatically inhibit DNA synthesis, as mediated through their interaction with DNA polymerase.
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PMID:Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine. 47 79

A regulatory protein for DNA polymerase alpha, responsive to noncomplementary deoxyribonucleoside triphosphates, has been isolated from calf thymus. The regulatory protein was separated from DNA polymerase alpha using Affi-Gel Blue and gel filtration. The regulatory protein had a molecular weight of approximately 70,000 as determined by gel filtration, and its activity was nondialyzable, heat labile, and abolished by pronase treatment. In the presence of regulatory protein, DNA polymerase alpha activity, measured by using polydeoxyadenylate-oligodeoxythymidylate as template primer, was inhibited by 2'-deoxyguanosine 5'-triphosphate in a parabolic-competitive fashion [Ki = 15 +/- 1 (S.E.) microM] and by 2'-deoxycytidine 5'-triphosphate in a linear-competitive manner (Ki = 162 +/- 23 microM). Neither the four natural ribonucleoside triphosphates nor 2'-deoxyadenosine 5'-triphosphate inhibited the DNA polymerase-regulatory protein system to any significant extent. The regulatory protein by itself had no effect on either DNA polymerase alpha activity or the Km for template primer. These results indicate that deoxyribonucleoside triphosphate pools may be involved in the regulation of cellular DNA synthesis through a direct effect on DNA polymerization.
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PMID:Isolation of a DNA polymerase alpha-associated regulatory protein from calf thymus. 49 66

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze extensive DNA synthesis on duplex phage T7 or PM2 DNA templates containing single strand breaks. A variety of physicochemical techniques have been used to characterize the DNA product synthesized in this reaction in the absence of ribonucleoside 5'-triphosphates. Pyknographic and sedimentation analyses reveal that all of the newly synthesized DNA is covalently attached to the template DNA. Analysis by electron microscopy shows the major portion of the product molecules synthesized on duplex T7 DNA templates to consist of a double-stranded branch attached to an intact template molecule. Using PM2 DNA templates, the predominant product consists of a double-stranded branch attached to the circular PM2 DNA template. Analyses of these product molecules indicate that DNA synthesis by the gene 4 protein and T7 DNA polymerase is initiated at single strand breaks in the duplex DNA and that synthesis is accompanied by extensive displacement of one of the parental strands. At later times in the reaction, a portion of the 3'-hydroxyl terminus of the newly synthesized DNA is displaced from the template by branch migration and is used as a primer by the DNA polymerase to copy the displaced 5' single-stranded parental strand to form a duplex branch.
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PMID:Gene 4 protein of bacteriophage T7. Characterization of the product synthesized by the T7 DNA polymerase and gene 4 protein in the absence of ribonucleoside 5'-triphosphates. 61 86

The enzyme which catalyses template independent synthesis of polydeoxynucleotides from deoxynucleoside diphosphates was separated from E. coli DNA polymerase I by DEAE-cellulose chromatography followed by ultrafiltration through the M-50 Amicon filter. The ultrafiltration data indicate that the molecular weight of the enzyme is not higher than 50,000. The enzyme is not able to use deoxynucleoside triphosphates, ribonucleoside di- or triphosphates as substrates for the polymerization. The reaction of template independent polymerization proceeds with a lag period varying from 2 to 20 hours (for different preparations of enzyme) and is activated by Mg2+ (the optimal concentration 1-2 . 10(-3) M). The pH optimum of the reaction is at 8.5. The optimal concentration of deoxyribonucleoside diphosphates is 10(-3) M, and its increase strongly inhibits polymerization. The enzyme was supposed to be called deoxynucleoside diphosphate: olygonucleotide deoxynucleotidyltransferase (catalyzing polymerization without template). The presence of the enzyme in the preparations of E. coli DNA-polymerase I can explain the ability of the latter to catalyze the untemplated synthesis of poly dG : poly dC.
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PMID:[Separation of the enzyme catalyzing polymerization of deoxyribonucleoside diphosphates from preparations of E. coli DNA-polymerase I]. 80 82

A protein factor, SF I, which stimulated DNA polymerase activity severalfold was purified from nuclei of sea urchin embryos by phase separation, ammonium sulfate fractionation, DNA-cellulose, CM-cellulose and hydroxyapatitecolumn chromatography and gel filtration. The molecular weight of SF I was about 220 000, the S20,W value was about 8.5 and the isoelectric point was determined to be pH 5.1. In the presence of SF I,V of the DNA-polymerizing reaction was increased and Km values for the substrates of this reaction were not changed. Addition of polyamines increased the rate of stimulation. ATP which was required for stimulation could be substituted by other ribonucleoside triphosphates. SF I, nuclear DNA polymerase and ATP seemed to form an active complex, and in the complex, ATP was found to have been converted to AMP and inorganic pyrophosphate.
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PMID:Stimulation of sea urchin DNA polymerase by protein factors. II. Formation of active complex in DNA polymerase reaction. 84 29

DNA alpha-polymerase has been partially purified from nuclei of cultured chic, fibroblasts and separated on phosphocellulose columns into two distinct activities designated DNA polymerases alpha(a) and alpha(b), respectively. The enzyme preparations were devoid of activities of DNA beta,gamma-polymerases terminal deoxyribonucleoside transferase, DNase, DNA-dependent RNA polymerase, and phosphatase. DNA polymerases alpha(a) and alpha(b) both having molecular weights of 160 000, constitute 35-50 and 65-50%, respectively, of the activity of alpha-polymerase in the nucleus. These enzymes differ in their requirements for maximal activity, their relative ability to copy oligo(dG)-poly(dC), their response to ribonucleoside triphosphates, and their kinetics of heat inactivation. When the properties of alpha polymerases derived from early or late passage cultures have been compared, no difference could be detected as a function of cell age in the specific activities of the polymerases in crude cell extracts, their chromatographic behavior on diethylaminoethylcellulose and phosphocellulose columns, and their relative abilities to utilize single deoxyribonucleoside triphosphates with activated DNA template. On the other hand, both enzymes become partially heat labile in aging cells. Also, the activity of DNA polymerase alpha(a) from young cells was stimulated by 2--10 mM adenosine or cytidine triphosphates, whereas the same enzyme from old cultures was inhibited by these agents. Conversely, these ribonucleoside triphosphates inhibited the activity of polymerase alpha(b) in young cells but slightly stimulated this enzyme derived from senescent fibroblasts. In addition, the relative ability of DNA polymerase alpha(a) to copy oligo(dG)-poly(dC) decreased in aged cells, whereas that of DNA polymerase alpha(b) increased. We have also observed significant differences in the effects of potassium chloride and N-ethylmaleimide on the activity of DNA polymerase alpha(a) from old cells as compared to young cells. These age-related alterations in the properties of the two avian DNA polymerases may reflect structural or conformational changes in these enzymes.
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PMID:Altered nuclear deoxyribonucleic acid alpha-polymerases in senescent cultured chick embryo fibroblasts. 98 31

2-Aza-1,N6-etheno-adenosine triphosphate (aza-epsilonATP), a fluorescent analog of adenosine triphosphate, significantly inhibits polyadenylate [poly(A)] polymerase of bovine lymphosarcoma and calf thymus, with 50% inhibition at 200 muM (in the presence of an equal concentration of adenosine triphosphate). Calf thymus RNA polymerases II and III are inhibited 32 and 20%, respectively, by a 3.8-fold excess of aza-epsilonATP; DNA polymerase alpha is not inhibited. The inhibition of poly(A) polymerase by aza-epsilonATP appears to be competitive with adenosine triphosphate; incorporation of aza-epsilonATP is not observed. Polymers of 2-aza 1,N6-etheno-adenosine monophosphate are used as primers, but pootly. 1,N-Etheno-adenosine triphosphate and 9-beta-D-arabinofuranosyladenine triphosphate are poor inhibitors of poly(A) polymerase; adenosine diphosphate is ineffective. Deoxyadenosine triphosphate inhibits to the same extent as aza-epsilonATP, while other naturally occurring nucleotides inhibit poly(A) polymerase to varying degrees, with deoxynucleoside triphosphates more potent than ribonucleoside triphosphates. Inhibition of poly(A) polymerase by naturally occurring nucleoside triphosphates suggests that nucleotides may regulate the enzyme in vivo; inhibition by the fluorescent analog aza-epsilonATP suggests that this compound may be useful in elucidating poly(A) metabolism in both normal and neoplastic cells.
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PMID:Inhibition of mammalian polyadenylate polymerase by 2-aza-1,N6-etheno-adenosine triphosphate. 98 43

The protein responsible for the initiation of conversion of single-stranded phage G4 DNA to the duplex replicative form has been purified approximately 3000-fold and identified with Escherichia coli dnaG gene product. The protein is a rifampicin-resistant RNA polymerase of approximately 64,000 daltons. It catalyzes the incorporation of the four ribonucleoside triphosphates into an oligoribonucleotide, using as template the single-stranded DNA coated with the DNA unwinding protein of E. coli. An RNA transcript of a unique region of the chromosome can serve as a primer by covalent extension by DNA polymerase III holoenzyme to form a nearly full-length linear complementary strand. A similar role for the dnaG protein in the initiation of nascent (Okazaki) fragments in replication of the host chromosome is discussed.
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PMID:dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. 109 46


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