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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 effect of heparin on DNA synthesis was compared between replicative DNA synthesis and unscheduled DNA synthesis. Replicative DNA synthesis in permeable cells or nuclei prepared from rapidly growing mouse ascites sarcoma cells was inhibited by heparin. Unscheduled DNA synthesis in nuclei isolated from normal rat liver or from mouse ascites sarcoma cells in stationary phase was stimulated by heparin at low concentrations but inhibited by high heparin concentrations. DNA polymerase activity assayed with activated calf thymus DNA and DNA polymerase alpha purified partially from mouse ascites sarcoma cells was inhibited with either calf thymus histones or heparin. DNA synthesis inhibited with histones was partially reactivated by heparin. Replicative DNA synthesis in permeable cells was inhibited by adding histones to the assay mixture, and the inhibited DNA synthesis was partially reactivated by low concentrations of heparin. These results indicated that the replicated sites (or replication machinery) in permeable cells or nuclei were largely unrestricted by histones and that heparin inhibition of replicative DNA synthesis was due to the direct inhibitory interaction of heparin with some essential component(s), such as DNA polymerase, of replication machinery.
Biochim Biophys Acta 1977 Dec 14
PMID:Differential effects of heparin on replicative DNA synthesis and unscheduled DNA synthesis. 92 8

Ataxia telangiectasia, Bloom's syndrome and normal fibroblasts were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase. It was found that an ataxia strain had substantially lower, and a Bloom's syndrome strain had slightly lower capacity than a normal strain; while the activities of apurinic site specific endonuclease in these extracts were comparable.
Biochim Biophys Acta 1977 Dec 14
PMID:DNA repair enzymes in ataxia telangiectasia and Bloom's syndrome fibroblasts. 92 14

Commercial preparations of guanosine 5'-triphosphate and deoxyguanosine 5'-triphosphate are contaminated with oligoribonucleotides 4 to 6 residues in length. The oligoribonucleotides can be separated from the nucleoside 5'-triphosphates by chromatography on DEAE-Sephadex A-25 or by gel filtration through Sephadex G-25. The oligoribonucleotides are effective primers for the DNA polymerase of bacteriophage T7 on the single-stranded circular DNAs of phage fd and phiX174; they are covalently attached to the 5' terminus of the newly synthesized DNA. The priming activity is specific; the oligoribonucleotides do not serve as primers for DNA polymerase I of Escherichia coli or for the DNA polymerase induced by phage T4.
J Biol Chem 1977 Dec 10
PMID:Priming of deoxyribonucleic acid synthesis on phage fd and phiX174 templates by oligoribonucleotides contaminating nucleoside 5'-triphosphates. 92 8

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.
Cancer Res 1976 Dec
PMID:Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver. 100 Apr 98

Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.
J Biol Chem 1976 Dec 25
PMID:Nuclear location of mammalian DNA polymerase activities. 100 14

Adenovirus 12 (Ad12) (Huie) (highly oncogenic group A) readily induces tumors in newborn rodents. Since Ad12 is isolated from human fecal samples, we investigated whether it plays a role in the etiology of human gastrointestinal cancer. If Ad12 is a causal agent of human cancer, then human tumors should contain Ad12 transforming genes, as indicated by studies of cells transformed in vitro and in vivo by oncogenic viruses. Ad12 DNA and the Ad12 transforming restriction fragment (EcoRI-C fragment, left 16% of the viral genome) were labeled in vitro to 10(7) to 4 X 10(8) cpm/mug by the nick translation reaction of DNA polymerase of Escherichia coli. The fidelity and sensitivity of these probes were established by (i) analysis of DNA from Ad12-transformed cells and from hamsters with tumors induced by Ad12, (ii) reconstruction experiments with added Ad12 DNA and EcoRI restriction fragments, and (iii) comparison of annealing characteristics with Ad12 probes labeled in vivo. With Ad12 [3H]DNA as probe, no viral DNA sequences were detected in 18 normal gastrointestinal tissues and 34 gastrointestinal tumors, including cancers of the colon, rectum, small intestine, and stomach, under conditions that would detect 0.1 copy of the Ad12 genome per tumor cell. Similar analyses of Ad12-transformed hamster cells and Ad12 primary hamster tumors indicated 6-18 copies per cell of over 90% of the viral genome. With the Ad12 EcoRI-C transforming fragment as probe, no hybridization was detected with 32 human gastrointestinal tumors and five normal tissues; this result excludes 1-2% of the Ad12 genome per tumor cell. Our date are strong evidence that Ad12 is not a major cause of human gastrointestinal cancer. The Ad12 transforming EcoRI-C fragment hybridized (50-68% efficiency) with other Ad12 isolates and with Ad18 and 31 (members of oncogenic group A), but not at all with 28 other human Ad serotypes (manuscript in preparation). Thus other group A members probably are also not involved in human gastrointestinal cancer. No viral DNA sequences were detected in 12 normal lungs and 22 lung tumors, suggesting that respiratory cancer does not involve an Ad12 etiology.
Proc Natl Acad Sci U S A 1976 Dec
PMID:Do highly oncogenic group A human adenoviruses cause human cancer? Analysis of human tumors for adenovirus 12 transforming DNA sequences. 107 16

A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques. The effects of Mn(II) bound at the active site of the enzyme on the longitudinal (T1p-1) and transverse (T2p-1) relaxation rates of the alpha, beta, and gamma phosphorus atoms and 5 protons of enzyme-bound thymidine 5'-triphosphate (dTTP) were measured at 40.5 MHz (31P), 100 and 220 MHz (1H). From frequency dependence of T1p-1, a correlation time of 7 X 10(-10) s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 A were calculated for the --CH3, H6, H'1, H'2, and H'4 protons. The calculated Mn(II) to phosphorus distances of 4.2, 4.8, and 3.2 A for the alpha, beta, and gamma phosphorus atoms indicates that Mn(II) corrdinates directly only with the gamma-phosphoryl group and that a puckered triphpsphate conformation exists for the enzyme-bound dTTP. This differs from the binary Mn(II)-dTTP complex in which alpha, beta, and gamma phosphoryl coordination occurs, and a thymine-deoxyribose torsion angly (chi) about the glycosidic bond of 40 degrees is detected. The eight manganese-substrate distances on the enzyme are fit by a unique Mn-dTTP conformation, with a torsion angle equal to 90 degrees, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B. Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing. Similarly, the binding of Mn-dATP to DNA polymerase I increased the distances from Mn(II) to the H2, H8, H'1, and H'4 protons of dATP but the adenine-deoxyribose torsion angle of 90 degrees was preserved. Such preorientation of substrates could facilitate incorporation of the complementary nucleotide. When positioned within the DNA structure, the conformation of enzyme-bound Mn-dTTP requires an inline nucleophilic attack on the alpha phosphorus with Mn(II) promoting pyrophosphate departure.
J Biol Chem 1975 Dec 10
PMID:Conformation of deoxynucleoside triphosphate substrates on DNA polymerase I from Escherichia coli as determined by nuclear magnetic relaxation. 110 9

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
Mol Gen Genet 1975 Dec 01
PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

A tenfold increase in the spontaneous reversion frequency of two of six lacZ frameshift mutations tested was observed in strains containing the following DNA polymerase I mutations--polA1, polA5, polA6, polAex1, res-3 and resA1. Reconstruction experiments indicated that this increase was not the result of a selective effect. Only a fourfold increase in frameshift mutations was found in strains containing a polA107 mutation. Both the polAex1 and polA107 mutations result in defective 5' to 3' exonuclease activity and do not affect polymerizing activity, but have different effects on frameshift mutation. A polA mutation on the chromosome induced frameshift mutations in a gene on an F episome. None of three auxotrophic mutations studied showed high frequency reversion in the presence of the polA1 or polA6 mutations.
Mol Gen Genet 1975 Dec 01
PMID:Increased spontaneous reversion of certain frameshift mutations in DNA polymerase I deficient strains of Escherichia coli. 110 18

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.
Biochim Biophys Acta 1975 Dec 04
PMID:Modified nucleotide polymers as inhibitors of DNA polymerases. 119 5


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