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)

Calcium elenolate, an antiviral agent which inhibits reverse transcriptases, inhibits the growth of chicken embryo fibroblast cells, as well as Echerichia coli and Bacillus subtilis strains. The drug in vitro inhibits E. coli deoxyribonucleic acid (DNA) polymerase II and DNA polymerase III holoenzyme, as well as several unrelated enzymes. The usual DNA polymerase assay components, with the exception of spermidine, have no effect on the observed inhibition. Inhibition of DNA polymerase II by the drug appears to be due to a direct and irreversible effect on the enzyme. However, DNA synthesis in E. coli is no more susceptible to the drug than is the increase in cell mass. These results suggest that calcium elenolate is an inhibitor of rather low specificity.
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PMID:Specificity of the antiviral agent calcium elenolate. 5 31

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA-dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180 000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt-free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the gamma-position at a maximal rate near 10(4) molecules per enzyme monomer per min at 35 degrees C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single-stranded, circular DNA of phages phiX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein phiX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA-dependent ATPase of molecular weight 69 000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.
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PMID:Enzymic unwinding of DNA. 1. Purification and characterization of a DNA-dependent ATPase from Escherichia coli. 13 22

Pancreatic DNase requires both Ca2+ and Mg2+ for its activity as measured by formation of an activated DNA template for in vitro DNA polymerase alpha assay and by the hyperchromic shift. Mn2+ can partially satisfy the Mg2+ requirement of the DNase for activation of DNA but the resulting template is only 50% as active in the DNA polymerase assay. When precautions are taken to avoid divalent ion contamination, pancreatic DNase is not active in the presence of Ca2+ or Mg2+ alone. analysis of the DNA by sucrose gradient centrifugation shows that only in the presence of Ca2+ plus Mg2+ or Mn2+ does pancreatic DNase produce extensive strand breaks in the DNA. The activated DNA template that yields maximal DNA polymerase activity is low molecular weight material of 30,000 to 50,000 daltons.
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PMID:Action of pancreatic DNase: requirements for activation of DNA as a template-primer for DNA polymerase. 41

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.
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PMID:Purification and properties of DNA polymerase-beta from guinea pig liver. 70 39

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.
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PMID:Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa. 74 11

It was recently reported (Lynch, W. E., Surrey, S., and Lieberman, I. (1975) J. Biol. Chem. 250, 8179-8183) that the extraction of regenerating rat liver in solutions of isotonic sucrose containing 4 mM CaCl2 leads to almost quantitative recovery of DNA polymerase alpha (Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., and Korn, D. (1975) Science 190, 401--402) activity in the purified nuclear compartment. Our application of this method to the isolation of the DNA polymerase activities activities from cultured human epithelial and lymphoblastoid cells has led to substantially different results. We have observed that the inclusion of Ca2+ in either isotonic sucrose or hypotonic aqueous extraction media leads to the irreversible inactivation of the majority, cytoplasmic fraction of DNA polymerase alpha activity and is without quantitative effect on the recovery of the nuclear fraction of this activity.
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PMID:Effect of calcium on the recovery and distribution of DNA polymerase alpha from cultured human cells. 86 13

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.
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PMID:Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos. 133 52

Investigations of mast cell biology have often used immortalized cultured cells which are continuously proliferating. In vivo, however, only 2% or fewer tissue mast cells are actively dividing. We used aphidicolin, an inhibitor of DNA polymerase to induce a proliferative arrest of murine mast cells characterized by an inhibition of cell division and thymidine incorporation, with accumulation of cells in G1 and early S phase of the cell cycle. Uridine incorporation and cell viability were not significantly impaired. DNA synthesis and cell division both resumed rapidly upon removal of the drug. Morphometric analysis demonstrated that cell size, granule size, and number of granules per cell were all increased in aphidicolin-treated cells. Proliferative arrest also produced a 14-fold increase in cellular histamine content, but did not alter the proteoglycans synthesized by the cell. The level of c-myc mRNA was reduced in aphidicolin-arrested cells, but returned to the level observed in untreated cells within 1 hr of removal of the drug. In contrast, the constitutive steady-state RNA levels of tumour necrosis factor-alpha (TNF-alpha), B2-microglobulin, actin, and the c-Ha-ras and c-fes protooncogenes were not altered. Aphidicolin-induced proliferative arrest did not prevent the induction of TNF-alpha, interleukin-6 (IL-6) and c-fos genes in response to calcium ionophore. Both the magnitude and induction kinetics of these messages were similar in aphidicolin-treated and untreated cells. We conclude that proliferative arrest results in morphological and biochemical changes suggestive of cellular maturation, but inhibition of cell division alone is not sufficient to alter mast cell phenotype. Although optimal c-myc expression appears to require active proliferation, cytokine gene induction can occur in non-dividing cells. These data suggest that the proliferative quiescence of in vivo mast cells should not preclude their involvement in biological events via elaboration of multi-functional cytokines.
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PMID:Aphidicolin-induced proliferative arrest of murine mast cells: morphological and biochemical changes are not accompanied by alterations in cytokine gene induction. 138 41

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.
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PMID:Heteroduplex DNA formation is associated with replication and recombination in poxvirus-infected cells. 165 5

We have used an in vitro assay to study the induction of DNA synthesis by cytoplasmic extracts from the actively growing cell line Molt 4 in nuclei isolated from quiescent human lymphocytes. The TTP incorporation which takes place in these nuclei has been shown to be inhibitable by serine protease inhibitors, particularly aprotinin. This DNA synthesis has also been proposed to reflect the initiation of true DNA replication; however, we find evidence that much, if not most, of this incorporation is due to nonreplicative synthesis initiated on primer templates formed by calcium-dependent activation of the nuclear chromatin substrate. The principal DNA polymerase supplied by the Molt 4 extract appears to be polymerase alpha and the results show that the activated chromatin is a substrate for purified bacterial DNA polymerases. DNA synthesis is significantly enhanced by preincubation at 37 degrees C in the presence of calcium, and the almost complete inhibition of DNA synthesis induced by extracts or bacterial polymerases in the presence of T4 ligase suggests that this chromatin activation involves calcium-dependent endonucleases. Nevertheless, DNA synthesis in the isolated nuclei, with both Molt 4 extracts and bacterial polymerases, is substantially inhibited by addition of serine protease inhibitors, with aprotinin the most potent of those tested on a molar basis. Thus, the results suggest that specific proteolytic activity is required before nicked or damaged nuclear DNA can serve as an acceptable substrate for DNA polymerase activity.
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PMID:DNA synthesis in isolated resting nuclei: evidence for protease-dependent nonreplicative nucleotide incorporation. 169 99


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