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)

Using initiated poly(dG,O6-RdG) and poly(dA,O6-RdG) polynucleotides as templates for DNA polymerase I in vitro the promutagenic potential of O6-MeG and O6-EtG has been confirmed, together with the possibility of minor miscoding pathways for O6-RG. These lead to the incorporation of dAMP and dGMP, which could give rise to some of the limited number of transversions that have been observed arising from the action of alkylating agents. The results are compatible with the current knowledge of oncogenes, explaining the changes in base sequence that have been observed. The competition for the miscoding of O6-RG which leads to the incorporation of dCMP in addition to the expected dTMP is also shown. The relative amounts of these two nucleotides incorporated depend upon the concentrations of the dCTP and dTTP in the assay. The mutagenic efficiency of O6-MeG is constant at approx. 0.4 over a wide range of dTTP and dCTP concentrations and only increases when the dCTP in the assay ceases to saturate the polymerizing enzyme, indicating that the DNA polymerase I plays a role in determining the mutagenic efficiency of a modified base. Although the mutagenic efficiency of both O6-MeG and O6-EtG depends upon the relative concentrations. of dTTP and dCTP in the assay, a reduction in the concentration of dCTP can be more effective at increasing the mutagenic efficiency than a corresponding increase in the concentration of dTTP. These results indicate the importance of cellular dNTP pools in determining the cellular response to agents.
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PMID:The competitive miscoding of O6-methylguanine and O6-ethylguanine and the possible importance of cellular deoxynucleoside 5'-triphosphate pool sizes in mutagenesis and carcinogenesis. 394 34

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.
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PMID:Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine. 394 81

JLS-V9 mouse bone marrow cells were readily adapted to suspension culture, chronically infected with Rauscher leukemia virus (RLV), and subsequently grown in 7.5- and 14-liter New Brunswick fermentors. The suspension-type cell system can be modified to produce virus with clearly defined properties, such as high ribonucleic acid-dependent deoxyribonucleic acid polymerase (RDDP) activity, high particle count, and high infectious particle count. Biological and biophysical properties of suspension-produced RLV were not affected by concentration and purification employing continuous-flow and rate-zonal centrifugation procedures. The RDDP assay was standardized and showed a linear incorporation of (3)H-thymidine 5'-monophosphate ((3)H-TMP) up to 30 min. Further characterization indicated that a high percentage of (3)H-TMP incorporation was due to RDDP.
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PMID:Adaptation and infection of mouse bone marrow (JLS-V9) cells in suspension culture for production of Rauscher leukemia virus. 412 75

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.
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PMID:Thymidine kinase activity in cardiac muscle during embryomic and postnatal development. 437 15

The in vitro conversion of single-stranded DNA from bacteriophage fd to duplex structures depends on E. coli RNA polymerase, DNA polymerase III, riboand deoxyribonucleoside triphosphates, Mg(+2), spermidine or DNA-unwinding protein of E. coli, and two additional protein factors, referred to here as Factors I and II. These two factors are also essential for dTMP incorporation catalyzed by DNA polymerase III and dependent on poly(dA).oligo(dT) primer-template. In the latter reaction, there is an absolute dependency on ATP or dATP.
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PMID:Involvement of two protein factors and ATP in in vitro DNA synthesis catalyzed by DNA polymerase 3 of Escherichia coli. 458 95

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.
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PMID:Mechanism of replication of single-stranded PhiX174 DNA. VII. Circularization of the progeny viral strand. 459 Oct 49

Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J/m2, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with Micrococcus luteus correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf thymus DNA polymerase alpha but was recognized as a template by DNA polymerase beta. The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules od dNMP) per one correndonuclease incision. The length of the DNA polymerase beta product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg(2)=nd four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate. The average numbers of deoxynucleotides incorporated per one DNAase I incision or per one nonspecific break, measured in control samples, were equal, amounting to 0.3 dTMP molecule. This value corresponded to 1.2 dNMP molecule; in our opinion, this reflects contaminating nuclease activity of the system used. The present results testify to the ability of DNA polymerase beta to repair synthesis by the "patch and cut' mechanism.
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PMID:The DNA polymerase beta reaction with ultraviolet-irradiated DNA incised by correndonuclease. 625 Jun 19

Phosphonoformate inhibited the replication of Herpes simplex virus (HSV) type 1 and type 2 in culture. The concentration required to inhibit the replication of both types of virus by 2 logs at 28 h post-infection was approximately 150 microM. It was more potent than phosphonoacetate against the growth of both virus types. A virus mutant which is resistant to phosphonoacetate was cross-resistant to phosphonoformate. Arsonoacetate, at 300 microM, had no antivirus activity. Phosphonoformate also inhibited HeLa and KB cell growth; at a concentration of about 500 microM, cell growth was inhibited by 50%. The anti-cell growth effects of the drug were completely reversible. The antivirus effect of phosphonoformate was partially reversible, depending on the time and duration of exposure of infected cultures to the drug. To obtain the maximum antivirus effect, phosphonoformate had to be added within the first 3 h post-virus-infection and be continuously present for at least 18 h. Phosphonoformate, added at 0 h post-infection, suppressed the induction of virus-specific DNA polymerase and DNAase activities. dTMP incorporation into DNA was preferentially inhibited in nuclei isolated from infected cells compared to uninfected cells, and the degree of inhibition varied with the ionic strength of the assay. Phosphonoformate was a potent inhibitor of the purified HSV-1 and HSV-2 DNA polymerases, inhibiting DNA polymerase activity by 50% at a concentration of 3 microM and ionic strength of 0.2.
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PMID:Mode of action of phosphonoformate as an anti-herpes simplex virus agent. 626 Jan 89

DNA polymerase delta from rabbit bone marrow has an associated 3'-5'-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 A by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase delta. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase delta has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase b aggregates to molecular weight species of 300000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, delta is a single polypeptide of 122000 apparent molecular weight. The DNA polymerase incorporates between 250000 and 300000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-2 at 37 degrees C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase alpha, inhibits both the DNA polymerase and 3'-5'-exonuclease activities of delta. DNA polymerase alpha from rabbit bone marrow does not share a common subunit with delta. Therefore, aphidicolin binding is not specific for alpha, and conclusions based upon the supposition that it is must be reconsidered.
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PMID:DNA polymerase delta: one polypeptide, two activities. 628 2

A DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14,000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.
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PMID:Properties of a DNA-binding protein in a culture medium of thymus cells. 629 62


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