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PubMed1979

[Experiments towards the viral etiology of a preleukemic syndrome: osteomyelofibrosis (author's transl)].

The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and templateprimers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation velocity measurements of the purified enzymes gave values of 150 000, 40 000, 100 000 and 70 000 daltons for DNA polymerase-alpha, DNA polymerase-beta, and DNA polymerase-gamma and the reverse transcriptase respectively. The purified reverse transcriptase was specifically inhibited by antisera to the reverse transcriptases of the two primate viruses, SiSV and GaLV. Antisera raised against the myelofibrotic spleen reverse transcriptase inhibited the homologous enzyme and also the reverse transcriptase from SiSV and GaLV. DNA polymerases alpha, beta and gamma from the same spleen were not inhibited by the antisera. These results constitute the first indication of a possible retroviral etiology for myelofibrotic syndrome. Since SiSV and GaLV are exogenous to all primates the results indicate that this polymerase was acquired and the results are most simply interpreted as indicating that virus related to the SiSV-GaLV group is present in man.

Child↗
PubMed1978

Inhibition of activities of DNA polymerase alpha, beta, gamma, and reverse transcriptase of L1210 cells by phosphonoacetic acid.

Phosphonoacetic acid has been shown to suppress replication of DNA tumor viruses by inhibiting the activity of virus-induced DNA polymerase and consequently viral DNA synthesis. We now have evidence to show that phosphonoacetic acid inhibits also the cellular DNA polymerases alpha, beta, and gamma of L1210 cells as well as reverse transcriptases of two type C viruses. Particularly, the DNA polymerase alpha is just as sensitive as the herpes virus induced DNA polymerase. The DNA polymerases beta and gamma required seven times more phosphonoacetic acid for a 50% inhibition of their activities. Phosphonoacetic acid inhibited the activities of the reverse transcriptase and terminal deoxyribonucleotidyltransferase only at higher concentrations. Kinetic analysis with the DNA polymerase alpha showed that the compound is a non-competitive inhibitor with respect to the substrates and uncompetitive inhibitor with the activated DNA template. Studies on time course of phosphonoacetic acid inhibition revealed that the compound is inhibitory even after the initiation of DNA synthesis. Phosphonoacetic acid also inhibited cell growth as well as the type C virus production; at concentrations above 50 microgram/ml, the inhibitory effect was more profound on the type C virus production than on cell growth.

Animals↗
PubMed1976

Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase.

Phosphonoacetate was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out. The results demonstrated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway. A detailed mechanism and rate equation for the inhibition were developed. For comparison to phosphonoacetate, pyrophosphate inhibition patterns and apparent inhibition constants were determined. Twelve analogues of phosphonoacetate were tested as inhibitors of the herpesvirus of turkey-induced DNA polymerase. At the concentrations tested, only one, 2-phosphonopropionate, was an inhibitor. The apparent inhibition constant for it was about 50 times greater than the corresponding apparent inhibition constant for phosphonoacetate. DNA polymerase alpha of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phosphonoacetate. The apparent inhibition constants for the alpha polymerase were about 10-20 times greater than the corresponding inhibition constants for the herpesvirus-induced DNA polymerase. Duck DNA polymerase beta, Escherichia coli DNA polymerase I, and avian myeloblastosis virus reverse transcriptase were not inhibited by phosphonoacetate.

Acetates↗
PubMed1978

DNA polymerase alpha and beta in the California urchin.

DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species.

Animals↗
PubMed1976

Ehrlich ascites tumor (EAT) chalone effects on nascent DNA synthesis and DNA polymerase alpha and beta.

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of 3H-thymidine (3H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50-200 mug/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude alpha- and beta-polymerase activities were inhibited. Crude DNA polymerase for C3H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or 'gapped' DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting alpha- and beta-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating the DNA ligase is not inhibited.

Animals↗
PubMed1979

DNA polymerase alpha, beta and gamma activities in ultraviolet irradiated CV-1 monkey cells.

To determine the possible role of DNA polymerase alpha, beta and gamma during the repair period following ultraviolet (lambda max : 254 nm) irradiation of monkey CV-1 cells, we measured the three enzymatic activities by using specific tests, either in crude extracts or after fractionation by sucrose gradient (5--20%) centrifugation at high salt concentration. When compared to the unirradiated control, we could not detect any significant variation in the levels of activity of DNA polymerases alpha, beta and gamma at any time (0, 12 to 48 h) after ultraviolet irradiation of the cells with doses ranging from 9 to 52.5 J.m-2.

Animals↗
PubMed1979

Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.

Adenoviruses, Human↗
PubMed1977

DNA polymerase-beta from the nuclear fraction of sea urchin embryos: characterization of the purified enzyme.

Approximately 2,500-fold purifications of DNA polymerase-beta from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus, was performed. The enzyme preparation, which was devoid of DNase and terminal deoxynucleotidyl transferase as contaminants, showed a sedimentation constant of 3.0 S in a sucrose density gradient, a molecular weight of 50,000 by gel filtration, and an isoelectric point of pH 8.1. The enzyme activity was resistant to sulfhydryl group inhibitors. Its optimal pH was 9.0-9.5 in Tris-maleate buffer and 10.0 in glycine buffer. The optimal NaCl concentration for the activity was 30-60 mM and about half of the activity remained at 0.4 M NaCl. As a template-primer, the enzyme preferred synthetic homopolymers to activated DNA. The order of this preference was as follows; poly (dA)-oligo (dT)12-18 greater than poly (rA)-oligo (dT)12-18 greater than activated DNA. The above results indicate that the enzyme corresponds to DNA polymerase-beta from vertebrate cells.

Animals↗
PubMed1978

DNA polymerases alpha beta and gamma in inherited diseases affecting DNA repair.

Fibroblasts derived from patients with diseases affecting DNA repair processes, such as Xeroderma Pigmentosum (classical and variant), Fanconi's anemia, Bloom's syndrome, Ataxia Telangiectasica, Progeria and Werner's syndrome, were assayed for the three DNA polymerases. The specific activities of these enzymes were found within the limits observed in normal human fibroblasts. Also the sedimentation properties of the three polymerases were unaltered.

Anemia, Aplastic↗
PubMed1978

Purification and properties of DNA polymerase-beta from guinea pig liver.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.

Animals↗
PubMed1979

Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus.

DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli. Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10. The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used. The apparent Km values for dUTP were very similar to those for dTTP. Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers.

Animals↗
PubMed1976

DNA polymerases of murine LBN/b leukemic cells.

In the post-mitochondrial fraction of murine LBN/b leukemic cells, four fractions with DNA polymerase activity (I, II, III, IV) were found. On the basis of ion exchanger affinity and poly(A), poly(C) and poly(Cm) replication ability, fraction I was classified as RNA-directed DNA polymerase of viral origin. On the basis of the differences in the ion exchanger affinity, molecular weight, template requirement, pH-dependence of enzymatic activity and NaCl concentration, divalent ion requirements and susceptibility to N-ethylmaleimide inhibition, fractions II, III and IV were classified as DNA-directed DNA polymerases beta, alpha and gamma, respectively. Three fractions, i.e. reverse transcriptase, and DNA-directed DNA polymerases beta and gamma, were found to incorporate dTMP on a poly(A)-oligo(dT) template-primer. Despite the similarity of the reaction of DNA polymerases beta and gamma with poly(A)-oligo(dT), some other properties of these enzymes suggest that they represent distinct enzymatic entities.

Animals↗
PubMed1976

Fluctuation in activity of the molecular forms of cellular DNA polymerase during infection by SV40.

Infection of BSC-1 cells by SV40 brings about an increase of 7--11-fold in DNA polymerase activity, found in the nuclei and cytoplasm, respectively. The overall ratio between activites of DNA polymerase beta (3.1S) and DNA polymerase alpha (5.5S) remains fairly constant throughout infection. However,there is a large increase in DNA polymerase alpha2 (7.1S) in the cytoplasm, and its appearance in the nuclei late in infection. The addition of 1 M NaCl to infected cytoplasm,causes an aggregation of DNA polymerase alpha into a higher sedimenting form (9.8S), termed DNA polymerase alpha3. DNA polymerase alpha1, alpha2 and alpha3 are different molecular forms of the same enzyme, as can be seen by their similar inhibition by N-ethyl-maleimide, heparin and NaCl. However, this new activity, alpha3, is stimulated by dithiothreitol to a greater extent at pH 9.30 than at pH 7.94. The conformational changes induced in DNA polymerase and its increase in activity during infection with SV40 are discussed.

Cell Line↗
PubMed1976

Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes.

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.

Animals↗
PubMed1978

DNA-dependent DNA polymerase species in male germ cells of the mouse.

Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as alpha and beta DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase beta only. A quantitative analysis showed that the activity of the DNA polymerase beta reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.

Animals↗
PubMed1978

Arabinosyl nucleosides. XIII. Influence of arabinofuranosylthymine on DNA-, RNA- and protein-synthesizing systems in vitro.

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.

Arabinonucleosides↗
PubMed1978

Association of DNA polymerase with nucleosomes from mammalian cell chromatin.

More than half of the DNA polymerase beta in mouse ascites cell chromatin was found to be associated with monomeric nucleosomal particles (produced by micrococcal nuclease treatment of chromatin). Almost all nuclear DNA polymerase activity in lymphocytes was found to be associated with nucleosomes. The nucleosome-associated enzyme was mainly DNA polymerase beta in chromatin from resting and mainly DNA polymerase alpha in chromatin from concanavalin-A-stimulated lymphocytes.

Animals↗