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 production of immunoreactive somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) by the established cell line derived from a human lung carcinoma CALU-6 has been evidenced in the serum-free medium in increasing concentrations as a function of the incubation time. Gel filtration in acid conditions of cell-conditioned medium collected after 72 h showed peaks of immunoreactive Sm-C/IGF-I in the elution volume corresponding to the molecular weight of the synthetic Sm-C/IGF-I, and in the high molecular weight region, where specific binding sites for Sm-C/IGF-I could be also demonstrated. These results indicate that this established cell line produces high amounts of immunoreactive Sm-C/IGF-I and of Sm-C/IGF-I carrier protein. The pooled fractions corresponding to the molecular weight of synthetic Sm-C/IGF-I showed a competitive binding curve parallel to the standard in the Sm-C/IGF-I RIA system, and a mitogenic activity on cells from the same line similar to the one observed using two different pure Sm-C/IGF-I preparations, obtained by chemical synthesis or by DNA recombinant technology. When a monoclonal antibody (sm-1.2) raised against Sm-C/IGF-I was added into the medium, the mitogenic effect observed by both synthetic and cell-derived Sm-C/IGF-I peptide was completely abolished; the monoclonal antibody also partially inhibited the effect of 10% fetal calf serum and the thymidine incorporation observed in serum-free medium without growth factors. In serum-free medium the monoclonal antibody produced a 45% reduction of cells in S phase by thymidine labeling index without modification of the growth fraction as determined by primer-dependent alpha-DNA polymerase labeling index. In conclusion it seems that Sm-C/IGF-I has a critical role in the autocrine stimulation of the replication of this cell line.
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PMID:Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor I on an established human lung cancer cell line. 337 14

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.
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PMID:Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization. 340 61

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).
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PMID:[DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation]. 408 23

Rauscher leukemia virus deoxyribonucleic acid polymerase is reversibly inactivated by 6 m guanidine-hydrochloride. Gel filtration in 6 m guanidine-hydrochloride reveals that the viral deoxyribonucleic acid polymerase consists of a single polypeptide chain of approximately 70,000 molecular weight.
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PMID:Reversible inactivation of the deoxyribonucleic acid polymerase of Rauscher leukemia virus. 411 52

varphiX174 RF (replicative form) II DNA, labeled in vivo with [methyl-(3)H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA(+) cells during RF replication. [(32)P]dCMP was incorporated into the gaps present in the RF II DNA with [alpha-(32)P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated (32)P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the (32)P-labeled fragments. Gel electrophoresis of the products formed by digestion of the (32)P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA(+) cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during varphiX RF replication.
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PMID:Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication. 452 6

Metronidazole (10 micrograms/ml) was rapidly bactericidal when added to log-phase cultures of Bacteroides fragilis strain 2624. DNA synthesis stopped immediately after addition of metronidazole, whereas synthesis of RNA and protein continued at linear rates for at least 60 min. Gel electrophoresis of DNA extracted from metronidazole-treated cells revealed no nicking or strand breakage in either the 2.9- or 2.0-megadalton plasmid of strain 2624. Also, no degradation of chromosomal DNA was seen. DNA polymerase activity, measured in vitro, was not diminished by prior treatment of the cells with metronidazole. Thus, the primary action of metronidazole is a rapid inhibition of DNA replication. The DNA remains structurally intact, DNA polymerase activity is not directly affected, and cells retain metabolic activity, synthesizing RNA and protein at unaltered rates.
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PMID:Mechanism of action of metronidazole on Bacteroides fragilis. 619 96

Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.
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PMID:Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7. 627 92

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.
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PMID:Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine. 643 58

The procedures of isolation and purification of DNA polymerase alpha from mature eggs of the teleost fish Misgurnus fossilis (loach) are described. The two forms of DNA polymerase alpha were separated by chromatography on hydroxylapatite. The physical properties of the purified isoenzymes alpha 1 and alpha 2 were studied. Both forms of alpha-polymerase had the same values of the Stokes radius (63 A) and sedimentation coefficients (6.8 S). Gel filtration and sedimentation analyses revealed that the calculated values of molecular weight (M) and friction ratio (f/fo) for the isoenzymes are equal to 170 000 (M) and 1.72 (f/fo), respectively. The isoelectric point is equal to 5.7 for alpha 1-polymerase and 5.8 for alpha 2-polymerase after isoelectric focusing of the enzymes in polyacrylamide gels. It is concluded that DNA polymerase alpha from loach eggs is represented by two species of the acidic high molecular weight proteins with a prominent spatial asymmetry.
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PMID:[Isolation of two forms of DNA polymerase alpha from roe of loach. Physico-chemical properties of the isoenzymes]. 662 13

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71


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