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)

Three forms of DNA polymerase (alpha, beta and gamma) were separated from isolated rat myocardial cells on the basis of template, pH and ionic requirements, sensitivity to N-ethylmaleimide and position on sucrose gradients. Tri-iodothyronine administration (20mug/100g intraperitoneally) to 3-week-old rats resulted in selective stimulation of DNA polymerase-alpha (198+/-7.1 versus 102+/-5.8pmol of [(3)H]dTMP/30min per mg of protein in untreated controls, P<0.01), with no change in polymerases-beta and -gamma. [(3)H]Thymidine incorporation into myocardial DNA was also enhanced in tri-iodothyronine-treated neonatal rats (132+/-11.2 versus 53+/-4.1c.p.m./mug of DNA in controls, P<0.001). Increased incorporation was associated with an expansion of deoxyribonucleoside 5'-triphosphate pools, especially that of dTTP (24+/-1.6 versus 10+/-1.1pmol/mg of DNA, P<0.01). Neither DNA polymerase activities nor [(3)H]thymidine incorporation were changed in 6-month-old rats in response to tri-iodothyronine. Unstimulated adult myocardial cells had DNA polymerase activities comparable with those in 3-week-old animals, but significantly lower [(3)H]-thymidine incorporation and deoxyribonucleoside triphosphate concentrations. Enhancement of both DNA polymerase-alpha activity and [(3)H]thymidine incorporation in tri-iodothyronine-treated young rats was prevented by concomitant administration of either vinblastine (1mug/g) or daunomycin (2mug/g); actinomycin D (0.1mug/g) or cycloheximide (8mug/g), on the other hand, prevented the increase in [(3)H]thymidine incorporation, but not DNA polymerase-alpha activation. These results demonstrate an age-dependent stimulation of myocardial DNA replication by tri-iodothyronine and suggest an inter-relationship between DNA synthesis and subsequent entry into mitosis.
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PMID:Selective stimulation by tri-iodothyronine of myocardial deoxyribonucleic acid polymerase-alpha in neonatal rats. 48 6

The labelling of mouse DNA by nick translation with DNA polymerase I has been investigated with respect to the time of incubation, requirement for DNAase I, size of the product, and uniformity of labelling, and the hybridisability and stability of the resultant labelled probes. Total mouse DNA and reannealed unique mouse DNA sequences can be labelled by nick translation in the presence of [3H]dCTP and [3H]TTP to a specific activity of 7 . 10(6)--20 . 10(6) cpm/microgram DNA. The hybridisation characteristics of nick-translated whole DNA with an excess of unlabelled mouse-embryo driver DNA indicates that no preferential labelling of repetitive or unique DNA sequence classes occurs. In addition, the proportion of unique DNA sequences labelled by nick translation which hybridises with polyadenylated nuclear RNA from Friend cells is the same as that of unique DNA sequences isolated from cells labelled with [3H]thymidine in vivo, indicating that few (if any) of the unique DNA sequences are unrepresented in the nick-translated probe. Probes which contain [3H]dTMP are unstable, and show a considerable reduction in hybridisability over a period of 6 months at --20 degrees C. The decrease is accompanied by an increase in the number of mismatched sites in duplexes containing the labelled probe (as shown by thermal stability measurements of hybrid molecules) and a decrease in the rate of hybridisation of the probe with total mouse DNA. In contrast, DNA which is labelled with [3H]dCMP alone is stable, and does not show any decrease in hybridisability on prolonged storage.
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PMID:Nick translation of mammalian DNA. 57 Apr 19

Chromatin prepared from S phase hepatoma tissue culture (HTC) cell incorporates in vitro about 11-14 pmoles [3H]dTMP into DNA in 30 min. Single-stranded DNA added to this chromatin stimulates DNA synthesis more than 40-fold whereas activated DNA enhances it about 60-fold. By contrast, stimulation of DNA synthesis by activated DNA in a crude nuclear extract exceeds the stimulation exerted by denatured DNA by a factor of 7. Stimulation of DNA synthesis by denatured DNA is not due to stabilization of either the chromatin or the product of the endogenous reaction. On the other hand, we find that poly(dC) and poly (dT) enhance DNA synthesis by serving as templates which are copied by chromatin in a true complementary fashion. It seems therefore, that eukaryotic cell chromatin is able to copy single-stranded DNA at a high efficiency. Chromatin of G1 arrested cell copies exogenous templates at a considerably reduced rate. The enzyme responsible for the copying of denatured DNA is tentatively identified as DNA polymerase alpha on the basis of its sensitivity to sulfhydril group blocking, its requirements for ions and failure to copy the ribo strand of oligo(dT) poly(A).
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PMID:Highly efficient copying of single-stranded DNA by eukaryotic cell chromatin. 67 65

[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

In earlier reports we have suggested that bacteriophate T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes. This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics. The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna- mutants. Their activities were essentially unchanged compared to the wild-type phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP.
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PMID:Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides. 106 86

During the fractionation of various enzymes concerned with DNA synthesis from the postmicrosomal supernatant fraction of various tissues, DNA polymerace [EC 2.7.7.7], thymidine kinase [EC 2.7.1.75], dTMP kinase [EC 2.7.4.9], deoxycytidine kinase [EC 2.7.1.74], and deoxycytidine monophosphokinase (dCMP kinase) [EC 2.7.4.14] were found in the pellet fraction of postmicrosomal supernatant. Further, the uridine kinase [EC 2.7.1.48] and aspartate transcarbamylase [EC 2.1.3.2] activities of postmicrosomal supernatant from various tissues were also present in this pellet fraction. The activities of DNA polymerase, thymidine kinase, uridine kinase, and aspartate transcarbamylase from normal and regenerating rat liver, and Yoshida sarcoma were higher in the pellet fraction than in the supernatant. On the other hand, the activities of dTMP kinase, dCMP kinase, and orotidine-5'-phosphate decarboxylase [EC 4.1.1.23] were lower in the pellet fraction than in the supernatant. The pellet fractions of regenerating rat liver and Yoshida sarcoma showed a remarkable incorporation of various precursors (thymidine, dTMP, deoxycytidine, and dCMP) into DNA in the presence of a suitable DNA template, ATP and all four deoxynucleoside 5'-triphosphates for DNA synthesis. Normal adult rat liver catalyzed a much smaller incorporation of all these precursors, except for dCMP.
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PMID:Intracellular distribution of various enzymes concerned with DNA synthesis from normal and regenerating rat liver, and Yoshida sarcoma. 113 86

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1'-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a 'histone-nucleate' could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.
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PMID:Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity. 121 90

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr=180000-200000), that in its presence (0.6 M NaCl or 0.12 M KCl) being 6.3 S (Mr=80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisified by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) X (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) X (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. maydis.
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PMID:A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity. 124 75

We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase beta, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiency in vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral OriS or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the "cleansing" of the viral genome of uracil residues before the start of replication.
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PMID:Uracil in OriS of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication? 133 82

Deoxynucleoside analogs, AZT and/or ddN, are the therapeutic agents currently utilized to inhibit the human immunodeficiency virus (HIV) reverse transcriptase. The effects of their anabolic products, AZT-triphosphate (AZT-TP) and ddCTP on human cellular DNA metabolic processes were studied using highly purified, structurally and enzymatically defined forms of the two major human host DNA polymerases, alpha and beta, and compared to those of the reverse transcriptase purified from HIV viron. Human DNA polymerase alpha during processive DNA synthesis is able to incorporate AZT-monophosphate (AZT-MP) but not ddCMP into DNA, causing chain termination. During its initial encounter with a primer terminus, polymerase alpha is able to incorporate both AZT-MP and ddCMP into DNA chains. Polymerase beta is able to incorporate AZT-MP and ddCMP into DNA, causing chain termination in both modes of DNA synthesis. Steady state kinetic analyses demonstrate that polymerase alpha inserts one AZT-MP molecule into DNA for every 2500 dTMP molecules incorporated. Polymerase beta incorporates ddCMP with efficiency nearly equal to that of dCMP. HIV reverse transcriptase prefers to incorporate AZT-MP and ddCMP rather than dTMP and dCMP, respectively. The findings described here raise the concern that the capability of the two major host DNA polymerases to incorporate AZT-MP or ddCMP into DNA might cause adverse side effects on human DNA metabolism and mutation in the genomes of patients under long term continuous treatment with AZT and ddC.
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PMID:Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA. 140 Apr 58


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