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

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. 142 73

We have synthetised 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate (in short : rATP-DNP), a derivative of ATP which carries a dinitrophenyl group. We show that rATP-DNP is a substrate for calf thymus deoxynucleotidyl terminal transferase (EC 2.7.7.31) and E. coli DNA polymerase I (Kornberg polymerase EC 2.7.7.7.). It can therefore be incorporated into DNA molecules by elongation from 3' ends or by nick translation. The incorporated dinitrophenyl group can be recognized by specific antibodies which can then be detected by anti-antibodies coupled to an enzyme. DNP groups could also be introduced into DNA after enzymatic incorporation of 8-aminohexyl adenosine 5' triphosphate and reaction with 1-fluoro-2-4-dinitrobenzene. Thus, DNA molecules carrying DNP groups can ultimately be revealed by enzymatic coloured reactions. Potential uses of this enzymatic labelling as a substitute to the radioactive detection of nucleic acids, are discussed.
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PMID:Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses. 675 67

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.
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PMID:Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion. 1223 19