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)

This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.
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PMID:Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells. 44 99

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.
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PMID:Isolation of a stimulatory factor for nuclear DNA replication. 53 38

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.
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PMID:The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes. 60 24

Amoeba discoides nuclear protein partially purified by passage through Sephadex G-200 showed 3 high-mol.-wt. DNA polymerase activities which eluted in and just following the void volume. No low-mol.-wt (45,000 daltons) DNA polymerase beta activity was detected. Nuclear protein layered on 5--20% sucrose gradients also showed an absence of low-mol.-wt DNA polymerase beta. The void volume enzyme showed deoxyribonuclease activity, but no low-mol.-wt nuclease activity was detected.
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PMID:Absence of low molecular weight DNA polymerase activity from the nuclei of Amoeba discoides. 63 29

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.
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PMID:DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities. 64 18

Upon exposure to the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz[a]anthracene, which bind covalently to DNA, ether-permeabilized (nucleotide-permeable) Escherichia coli wild-type cells responded with DNA excision repair. This repair was missing in mutants carrying defects in genes uvrA, uvrB and uvrC, whereas it was present in uvrD and several rec mutants. Enzymic activities involved were identified by measuring repair polymerization and size reduction of denatured DNA. 1. An easily measurable effect in E. coli wild-type cells was carcinogen-induced repair polymerization. When initiated by N-acetoxy-N-2-acetylaminofluorene or 7-bromomethyl-benz[a]anthracene, it depended upon an ATP-requiring step; CTP, GTP or UTP did not substitute for ATP. DNA repair synthesis was inhibited by p-chloromercuribenzoate and quinacrine. In uvrA, uvrB and uvrC mutants no carcinogen-stimulated DNA synthesis could be detected, indicating that steps involved in pyrimidine dimer excision are also involved in chemorepair. In recA, recB and recC mutant cells, repair synthesis was stimulated by the carcinogens to a normal extent. This evidence excludes the ATP-dependent recB,C deoxyribonuclease and recA gene products as playing an important role in carcinogen-induced excision repair. polA1 cells showed drastically reduced levels of rapair polymerization, indicating that DNA polymerase I is the main polymerizing enzyme. 2. As determined by DNA size reduction in alkaline sucrose gradients, the arylalkylating carcinogens caused endonucleolytic cleavage of endogenous DNA in wild-type cells. This incision step was most effectively performed in the presence of ATP; UTP, CTP and GTP were only slightly effective. Incision was inhibited by p-chloromercuribenzoate and quinacrine. When exposed to the arylalkylating carcinogens, uvrA, uvrB and uvrC mutant cells did not perform the incision step in the presence of ATP, suggesting the involvement of the respective gene products in the initiation of chemorepair.
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PMID:Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz(a)anthracene. 76 31

A ribonuclease-sensitive DNA polymerase, which uses an endogenous template, is detectable in the 39,000 g supernatant of a rat thymus homogenate, and appears as a single peak of activity in the void volume after Sephadex G 150 or G 200 gel filtration chromatography. Native and "activated" DNA-dependent DNA polymerase activities coincide with the endogenous-templated polymerase activity. Treatment of the thymus extract with ribonuclease(s) prior to gel filtration chromatography yields two other peaks of activity in addition to the void volume peak. The appearance of the two lower molecular weight peaks of activity is accompanied by a concomitant decrease in the endogenous-templated activity. The effect of ribonuclease is specific and cannot be reproduced by a similar deoxyribonuclease treatment.
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PMID:DNA polymerase activity associated with endogenous template: release by ribonuclease treatment. 80 37

Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methanesulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methanesulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis. In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism. The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.
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PMID:Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damages in mycobacterium smegmatis. 84 85

The 3' leads to 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.
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PMID:A DNA polymerase from Ustilago maydis. Evidence of proof-reading by the associated 3' leads to 5' deoxyribonuclease activity. 89 48

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


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