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)

Hepatitis B core antigen (HBcAg) particles, approximately 27-28 nm in diameter and rho = 1.30-1.35 g/cm3, were purified from the liver of a chimpanzee experimentally infected with hepatitis B virus (HBV) while under cyclophosphamide treatment. The purified HBcAg particles incorporated radioactive deoxythymidine triphosphate. The product was precipitable by trichloroacetic acid and sensitive to DNase, but resistant to digestion by RNase. The reaction required four deoxyribonucleosise triphosphates- dATP, dCTP, dGTP and dTTP. Exogenous template did not enhance the reaction. From these findings, it was suggested that HBcAg particles purified from the HBV-infected chimpanzee liver contained DNA polymerase and endogenous DNA.
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PMID:Hepatitis B core particles with endogenous DNA polymerase activity from chimpanzee liver. 68 Nov 46

[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.
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PMID:Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP. 70 36

The natural metabolite of the sponge Cryptotethya crypta, arabinofuranosylthymine (araThd), is intracellularly phosphorylated to araTTP. The present study demonstrates that araTTP inhibits both isolated DNA polymerases alpha and the DNA polymerase beta from L5178y cells competitively with respect to the analogous substrate dTTP. The affinity of araTTP is higher to the DNA polymerase alpha than to the DNA polymerase beta. The activity of mammalian DNA-dependent RNA polymerases I, II and III as well as the incorporation rate of a protein cellfree system is not affected by high doses of araTTP.
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PMID:Arabinosyl nucleosides. XIII. Influence of arabinofuranosylthymine on DNA-, RNA- and protein-synthesizing systems in vitro. 70 84

A novel DNA polymerase, which could use both poly(rA) . oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocussing (pI = 6.5--7.0) or sucrose rate-zonal centrifugation (10--10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) . oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) . oligo(dT)-dependent activity differed from the previously described DNA polymerase gamma from other sources in the following ways: 1. The activity was inhibited by 100--300 mM KCl and and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26 degrees C than at 37 degrees C. 3. The Km value for dTTP was 2.6--3.0 . 10(-4) M, which is several hundred-fold greater than that of DNA polymerase gamma. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase alpha, except that it was less sensitive to N-ethylmaleimide and anti-alpha polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.
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PMID:10-S DNA polymerase from calf thymus which copies both poly(rA) . oligo(dT) and activated DNA. 71 38

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.
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PMID:Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand. 77 43

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.
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PMID:Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. 77 32

The direct measurement of ultraviolet light-stimulated DNA synthesis in the permeable Bacillus subtilis cells was performed. Bacillus subtilis spores germinated in the presence of chloramphenicol were treated with Brij 58 and irradiated with ultraviolet light, and (3H)dTTP was incorporated into these cells by the DNA polymerase assay system. Characteristics of the incorporation were distinct from those into spores germinated in the absence of chloramphenicol and treated with Brij 58, in the respect that the former incorporation did not require ATP and only partially depended on the presence of all four deoxyribonucleoside triphosphates. The incorporation of (3H)dTTP into DNA was confirmed by CsCl density gradient centrifugation. A DNA polymerase I-deficient strain, JBl 49(59) had no (3H)sTTP incorporating activity induced by ultraviolet light irradiation when the germinated spores were treated with Brij 58. Analysis of alkaline sucrose gradient centrifugation revealed that fragmented DNA caused by ultraviolet light irradiation was rejoined to the size of DNA of non-irradiated cells by incubating irradiated cells in the DNA polymerase assay mixture containing NAD+. The results also suggested that a machinery of DNA repair probably pre-existed in the spore.
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PMID:Deoxyribonucleic acid synthesis induced with ultraviolet light in Brij 58-treated Bacillus subtilis spores germinated in the presence of chloramphenicol. 80 21

Extracts of Bacillus subtilis contain a deoxyuridinetriphosphatase (dUTPase) activity with a molecular weight of approximately 48,000. The enzyme is maximally active at pH 8.5, being stimulated by Mg2+ and inhibited by EDTA. The enzyme is specific for dUTP among all the natural nucleotides tested, with an apparent Km for dUTP of 2 muM. Bacteriophage PBS2, whose DNA contains uracil instead of thymine, induces upon infection of B. subtilis a new 83,000-dalton protein which inhibits the host's dUTPase. The inhibitor acts immediately and reversibly in vitro to inhibit dUMP production from dUTP. The inhibitor's action is maximal in dUTPase assays performed at pH 6 to 7, and is minimal at pH 9.7. The inhibitor seems to form a higher molecular weight complex with the B. subtilis dUTPase. Increasing the pH of the medium for PBS2 infection from pH 7 to pH 8.85 caused a dramatic decrease in the synthesis of phage DNA and progeny phage. The newly synthesized DNA had an altered thymine/uracil ratio, being increased from less than 0.03 to greater than 1.0. We propose that infection at high pH prevents the PBS2-induced dUTPase inhibitor from blocking the B. subtilis dUTPase activity, thereby allowing the degradation of dUTP and the synthesis of dTTP (both of which are DNA polymerase substrates), so that thymine replaces some of the uracil normally found in PBS2 DNA.
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PMID:Bacillus subtilis deoxyuridinetriphosphatase and its bacteriophage PBS2-induced inhibitor. 81 Apr 87

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.
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PMID:Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis. 82 70

1. DNA polymerase from the mesophile Bacillus licheniformis and the thermophile Bacillus stearothermophilus has been used to study the replication of poly(dA-dT)-poly(dA-dT) and poly(dC)-poly(dG) templates at 37, 45, and 55 degrees C. 2. Incorporation of non-complementary deoxyribonucleoside triphosphates (misincorporation) occurred with both enzymes and both templates. Non-specific incorporation (de novo polynucleotide synthesis, random attachment to existing strands, and tritium exchange of nucleotides) accounted for, at most, a small fraction of the total observed misincorporation. The error rates at 37 degrees C for the complete system were as follows:: B. licheniformis: dATP, 1/61; dCTP, 1/830; dGTP, 1/360; dTTP, 1/65; B. stearothermophilus: dATP, 1/68; dCTP, 1/1430; dGTP, 1/440; dTTP, 1/67. For both organisms, the error rate for dCTP and dGTP was independent of incubation temperature; the error rate for dATP and dTTP was 5-50-fold greater than that for dCTP or dGTP and increased significantly from 37 to 55 degrees C. 3. The ratio of dATP to dTTP incorporation with the poly(dA-dT)-poly-(dA-dT) template was independent of temperature and close to unity. The ratio of dCTP to dGTP incorporation with the poly(dC)-poly(dG) template decreased from approx. 0.2 to 0.05 for the mesophile and from approx. 0.06 to 0.03 for the thermophile as the temperature increased from 37 to 55 degrees C.
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PMID:DNA polymerase from mesophilic and thermophilic bacteria. III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus licheniformis and Bacillus stearothermophilus. 84 45


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