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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)

DNA synthesis has been studied in nuclei isolated from phytohaemagglutinin-stimulated lymphocytes from normal subjects and patients with megaloblastic anaemia. Lymphocytes were incubated for 72 h, nuclei isolated and incorporation of tritiated deoxythymidine triphosphate ([3H]TTP) into DNA measured, usually over a 10 min incubation period. Preincubation of normal phytohaemagglutinin-stimulated lymphocytes with methotrexate (1 - 10(-5) M, 48--72 h), 5-fluorouracil (1 - 10(-6) M, 70--72 h), and 1-beta-D-arabinofuranosyl cytosine (cytosine arabinoside) (4 - 10(-5) M, 71--72 h) caused a mean rise in [3H]TTP incorporation of 1.7 (P less than 0.01), 1.7 (P less than 0.05) and 2.4 (P less than 0.0025) fold, respectively. Hydroxyurea (3 - 10(-4) M, 48--72 h) in two experiments caused a mean increase of 1.6 fold. Untreated vitamin B-12- and folate-deficient cells showed a 2.0-fold (P less than 0.05) increase above the incorporation when the deficiencies were corrected by addition of vitamin B-12 and folic acid between 0 and 72 h in vitro. The mean percentages of the incorporation due to ATP-independent synthesis in nuclei from normal untreated cells, 5-fluorouracil-treated, cytosine arabinoside treated and vitamin B-12- or folate-deficient cells were 56 +/- 7% S.E., 41 +/- 7%, 84 +/- 3% and 28 +/- 6%, respectively. 5-Fluorouracil caused a two-fold increase in the cytoplasmic fraction of DNA polymerase when added to phytohaemagglutinin-stimulated lymphocytes between 48 and 72 h of culture but had no significant effect when added between 70 and 72 h.
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PMID:DNA synthesis in isolated lymphocyte nuclei. Effects of megaloblastic anaemia due to folate or vitamin B-12 deficiency or antimetabolite drugs. 88 15

Treatment with methylglyoxal bis(guanylhydrazone), a specific inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), suppressed the phytohemagglutinin-induction of [3H]thymidine uptake by guinea pig lymphocytes. The kinetics of [3H]thymidine uptake revealed that the Km value for thymidine was not changed, but the V value was markedly lowered by the methylglyoxal bis(guanylhydrazone) treatment. The induction of ATP: thymidine 5'-phosphotransferase (EC 2.7.1.75) (thymidine kinase) activity by phytohemagglutinin was suppressed to about the same extent as the induction of thymidine uptake. These suppressions were dependent on the methylglyoxal bis(guanylhydrazone) doses and on duration of the methylglyoxal bis(guanylhydrazone) treatment. Analysis of [3H]thymidine labelled compounds of the acid-soluble fraction showed that conversion of thymidine to thymidine 5'-triphosphate was inhibited by the methylglyoxal bis(guanylhydrazone) treatment. DNA polymerase activity was less inhibited by the methylglyoxal bis(guanylhydrazone) treatment in comparison with the methylglyoxal bis(guanylhydrazone) inhibition of thymidine uptake by whole cells. These results strongly suggested that blocking of polyamine accumulation by the methylglyoxal bis(guanylhydrazone) treatment influenced phytohemagglutinin induction of thymidine phosphorylation, resulting in a decrease of thymidine incorporation into DNA.
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PMID:Suppression of phytohemagglutinin-induction of thymidine uptake in guinea pig lymphocytes by methylglyoxal bis(guanylhydrazone) treatment. 91 40

Enzymes of DNA synthesis, thymidine kinase (ATP-thymidine-5'-phospho-transferase, EC 2.7.1.21), DNA polymerase (EC 2.7.7.7) and nuclease activities were investigated in isolated purified nuclei of swine aorta. Thymidine kinase which is detectable in these nuclei can be stimulated by the addition of phospholipase C. DNA polymerase activity of isolated nuclei is strongly dependent on addition of an exogenous template; the preferred template is activated DNA. The activity in the absence of an added template is very low except when labelled dCTP is used as the precursor. This incorporation of labelled dCTP does not require the addition of the other three triphosphates, and under these conditions, dCTP seems to be incorporated into what may be a homopolymer. As with other tissues, solubilized preparations of aortic nuclei have two DNA polymerase activities which also prefer activated DNA template. There is no detectable endonuclease in aortic nuclei.
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PMID:Enzymes of DNA synthesis in isolated nuclei of swin aorta. 94 21

The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.
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PMID:Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei. 94 95

Although DNA polymerase-alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-alpha (and DNA polymerase-beta) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-alpha is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 5.7.1.75) is found primarily in the enucleated cell fragments (cytoplasts). The enucleation procedure used in this work should be of general use for intracellular location studies.
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PMID:Intracellular localization of mouse DNA polymerase-alpha. 106 93

Addition of an extract prepared from a proliferating cell line to nuclei isolated from resting tissues such as frog liver and spleen resulted in the stimulation of DNA synthesis as assayed by [3H]dTTP incorporation. This stimulated incorporation of [3H]dTTP required ATP and depended on Mg2+ and deoxynucleoside triphosphates. Pulse-chase experiments showed that the synthesis of DNA in this system was discontinuous, resulting in the appearance of approximately 4S fragments and their ligation to yield higher molecular weight DNA. In addition, electron microscopic analysis of the DNA molecules from the reaction mixture showed that the frequency of replication "eyes" in the extract-stimulated reaction was 10-fold higher than that observed in controls. All of these results strongly suggest that the extract stimulated initiation of DNA replication in the chromatin of normally resting cells. Preliminary characterization by dialysis, heating, and enzyme treatments indicated that the activity is associated with one or more proteins of high molecular weight (greater than 50,000). Comparison of the levels of stimulatory activity in extracts from various mammalian and avian sources showed that the activity was present in cells proliferating either in vivo or in tissue culture. In contrast, extracts from normally resting tissues and cells had no activity. The level of activity present did not appear to be directly related to the levels of DNA polymerase. These results suggest a use for this system in studying regulation of the initiation of DNA synthesis and control of the various phases of the cell cycle.
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PMID:Initiation of replication in chromosomal DNA induced by extracts from proliferating cells. 106 72

Treatment of HeLa cells with a hypotonic buffer solution makes them permeable to nucleotides. Cells which are in S-phase at the time of treatment continue to synthesize DNA when supplied with the four deoxyriboside triphosphates, ATP, Mg2+, and the proper ionic environment. DNA replication extends from sites which were active in the cells prior to treatment. The product is confined to the nucleus and is sensitive to deoxyribonuclease. Under optimum conditions, up to 5% of the HeLa genome can be replicated from exogenous nucleotides. In synchronized cultures the level of DNA replicase activity, as measured in permeable cells at different points in the cell cycle, correlates with the rate of [14C] thymidine incorporation measured in the living, untreated cells.
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PMID:A permeable cell system for studying DNA replication in synchronized HeLa cells. 109 Mar 1

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented.
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PMID:DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli. 109 53

In crude extracts from Escherichia coli cells the ATP-dependent exonuclease V was found to be most active in converting double-stranded DNA into a suitable template for DNA polymerase. This phenomenon was studied in some detail with isolated exonuclease V and T7 DNA polymerase. We found that, at ATP concentrations arount 1 mM, the exonuclease produces a broad spectrum of DNA fragments. One class of fragments is largely single stranded with hydrogen-bonded small primer sequences. These structures allow the synthesis of remarkably homogeneous polynucleotide strands by T7 DNA polymerase.
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PMID:The mechanism of template activation by exonuclease V. 110 Mar 74

Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' leads to 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required.
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PMID:DNA repair synthesis dependent on the uvrA,B gene products in toluene-treated cells. 110 Jun 28


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