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)

The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma.
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PMID:Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma. 694 22

DNA polymerases alpha and gamma have been studied in cryptobiotic cysts and developing embryos and larvae of the brine shrimp Artemia. The two enzymes readily separate on Cibacron blue 3-GA Matrex gel. Assay requirements with activated DNA as primer-template are pH 8.0, 1 mM Mg2+, 50 mM K+ for DNA polymerase alpha and pH 8.4, 10 mM Mg2+, 80 mM K+ for DNA polymerase gamma. DNA polymerase alpha is inhibited by N-ethylmaleimide (94% and 100% at 1 mM and 10 mM respectively) and aphidicolin (96% at 60 microM). DNA polymerase gamma is also sensitive to N-ethylmaleimide (83% and 100% inhibition at 1 mM and 10 mM respectively) but is resistant to aphidicolin. 2',3'-Dideoxythymidine 5'-triphosphate (ddTTP) inhibits the gamma polymerase by 88% when in fivefold excess over dTTP whereas the alpha polymerase is unaffected by this compound. DNA polymerase alpha has a sedimentation coefficient of 7.6 S which is reduced to 6.2 S by a phenylmethylsulphonyl fluoride-sensitive proteinase. The gamma polymerase sediments at 8.3 S. No DNA polymerase beta activity could be detected. After the reinitiation of development both activities increased twofold up to 8 h (gamma polymerase) and 16 h (alpha polymerase), then declined before the onset of nuclear DNA replication after hatching. Thymidine kinase activity increased over 200-fold up to the time of replication. Analysis on Percoll density gradients of the intracellular distribution of both polymerases during development suggests that the changes in their activities may be due to migration from storage sites to replication complexes in the nuclei and mitochondria. The content of the mitochondrial DNA polymerase gamma in different batches of cysts may reflect the relative viabilities of the cysts.
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PMID:DNA polymerases alpha and gamma during pre-emergence and early larval development of Artemia. 715 4

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.
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PMID:Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase alpha and beta. Extraction, separation, characterization and changes during spermatogenesis. 719 42

In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.
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PMID:Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates. 723 28

Three forms of DNA polymerase, named enzymes A, B, and C, that preferred (rA)n x (dT)12-18 as a template-primer, were partially purified from an extract of rat spleen. Enzymes B and C, both sedimenting at 9S, appeared to correspond to DNA polymerase gamma. However, they differed in their behavior on phosphocellulose and DNA-cellulose column chromatographies, and in their optimum KCl and divalent cation requirements for activity. Enzyme A showed a unique property. Like DNA polymerase beta, it sedimented at 3.8S, was resistant to reagents blocking sulfhydryl groups, and was inhibited by phosphate, but it differed from DNA polymerase beta with respect to elution positions from DEAE-cellulose, phosphocellulose and DNA-cellulose columns, Km value (lower by one order of magnitude for dTTP), and template-primer preference. Enzyme A was found in the mitochondrial fraction, in which DNA polymerase beta was not detectable. Enzymes A and C were isolated from the nuclear fraction, but this fraction did not contain enzyme B. The cytosol contained only enzyme A. The mitochondrial fraction contained enzyme A and enzyme C-like polymerase. Enzyme B was obtained with enzymes A and C only by extraction of the whole cell homogenate. Enzyme B may be labile or may be an artificial form of DNA polymerase gamma formed during the purification procedures.
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PMID:Heterogeneous forms of poly(rA) . oligo(dT)-directed DNA polymerase activity from rat spleen. 724 Jan 27

DNA polymerase alpha (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase alpha, 6.5 S DNA polymerase alpha-1 and 6.5 S DNA polymerase alpha-2 (Masaki, S. and Yoshida, S. (1978) Biochim, Biophys. Acta 531, 74-88; Yoshida, S., Yamada, M., Masaki S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955-3958). Among these three, 10 S DNA polymerase alpha and 6.5 S DNA polymerase alpha-2 were found to copy efficiently poly(rA) . oligo(dT), a template-primer, which was thought to be specific for DNA polymerase gamma or beta. 6.5 S DNA polymerase alpha-1, however, could not use the ribopolymer as a template. The poly(rA) . oligo(dT)-dependent activities of DNA polymerase alpha species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase alpha, did not inhibit the poly(rA) . oligo(dT)-dependent activity. 2',3'-DideoxyTTP, a potent inhibitor of DNA polymerase beta or gamma, slightly inhibited the reactions with poly(rA) . oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent Km values for dTTP on poly(rA) . oligo(dT) template were 260 and 70 microM for 10 S alpha and 6.5 S alpha-2, respectively; these values were much higher than those obtained on activated DNA template (8-10 microM).
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PMID:Further characterization of a poly(rA) . oligo(dT)-dependent activity of multiple DNA polymerase alpha from calf thymus. 728 77

Mouse myeloma DNA polymerase gamma was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)n.(dT)12-18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of Mr = 47 000 changed proportionally with DNA polymerase gamma activity. A minor polypeptide of Mr = 140 000 also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by 125I-labeled peptide mapping indicates that the Mr 47 000 polypeptide in the mouse myeloma DNA polymerase gamma preparation is structurally related to the Mr 47 000 polypeptide of chick embryo DNA polymerase gamma (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002-7009). An antibody against chick embryo DNA polymerase gamma cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the Mr 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the Mr 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 per nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the 'expected' specific activity with respect to the Mr 47 000 polypeptide and the molecular weight (Mr = 188 000 on the assumption of a tetrameric structure of the Mr 47 000 polypeptide). Results indicate that the Mr 47 000 polypeptide is a component of the mouse myeloma DNA polymerase gamma.
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PMID:Identification of a polypeptide component of mouse myeloma DNA polymerase gamma. 728 87

Three DNA polymerases were partially purified from the embryos, liver and mitochondria of the loach Misgurnus fossilis by DEAE- and phosphocellulose chromatography and were identified as DNA polymerases alpha, beta and gamma. DNA polymerase alpha prefers the activated DNA as a template-primer and has a sedimentation value of 6.8S. The enzyme is stimulated by 50 mM potassium phosphate, KCl and, to a lesser extent, NaCl; has a pH optimum of 7.5 and is sensitive to N-ethylmaleimide. DNA polymerase beta also prefers the activated DNA as a template-primer but shows a sedimentation coefficient of 3.0 S. The enzyme is inhibited by salts and has a pH optimum of 8-9; its activity is rather resistant to N-ethylmaleimide. DNA polymerase gamma has a sedimentation value of 6.3S, shows a high activity on poly(A) . oligo(dT) in the presence of 100 mM potassium phosphate and a lesser activity in the presence of 150 mM KCl and NaCl. The enzyme has a pH optimum of 7.0.
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PMID:[Isolation and characterization of DNA polymerase alpha, beta and gamma from the cells of the loach (Misgurnus fossilis)]. 729 16

Thymidine triphosphate (TTP) and its halogenated analog bromodeoxyuridine triphosphate (BrdUTP) were compared in vitro as substrates for several prokaryotic and eukaryotic DNA polymerases to determine a possible enzymatic preference which might account for the reported finding of nonrandom patterns of incorporation of the analog in eukaryotic cellular DNA as well as help clarify the mechanism for drug-induced activation of latent retroviruses from animal cells. Following nucleotide competition reactions, no discriminatory utilization was detected from a mixture containing equimolar [3H]TTP and [alpha 32P]TTP for any of the polymerases. In contrast, when [3H]BrdUTP was mixed with an equal concentration of [alpha 32P]TTP, it was apparent that eukaryotic DNA polymerase gamma utilized more of the brominated analog triphosphate in preference to the unsubstituted compound. This increased affinity was confirmed by the differences in Km values. Furthermore, the selectivity of polymerase gamma was even more pronounced with the iodinated thymidine analog iododeoxyuridine triphosphate. On the other hand, polymerase gamma failed to discriminate as readily between equal concentrations of alpha 32P-labeled deoxycytidine triphosphate and 125I-labeled iododeoxycytidine triphosphate.
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PMID:Preferential utilization of bromodeoxyuridine and iododeoxyuridine triphosphates by DNA polymerase gamma in vitro. 729 97

The nature of the inhibitory effects of rifamycin derivative AF/013 (O-n-octyloxime of rifamycin SV) on DNA polymerases alpha and gamma were studied. Lineweaver-Burk analysis of the inhibition of DNA polymerases with respect to a substrate and template-primer showed a different mode of inhibition by AF/013 for each: the inhibition of DNA polymerase gamma was competitive with both dTTP and poly(rA)-oligo(dT), while that of DNA polymerase alpha was competitive with activated calf thymus DNA and non-competitive with dTTP. Further analysis of the competitive mode of the inhibition of DNA polymerase alpha, using poly(dT)-oligo(rA) as a template-primer, demonstrated that the primer molecule competed with AF/013. A change of effective divalent metal ion (Mn2+ in place of Mg2+) in the reaction mixture did not alter this competitive mode of inhibition with respect to the template-primer. The results of experiments to obtain further insight into the mechanism of drug-enzyme interaction suggest that AF/013 binds tightly to DNA polymerase alpha, and inhibits the process of chain elongation with DNA polymerases alpha and gamma.
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PMID:Inhibition of DNA polymerases alpha and gamma by rifamycin derivative AF/013. 733 11


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