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At least 19 recordsLinked to original sources
PubMed1979

Steady-state kinetics of mouse DNA polymerase beta.

DNA polymerase beta from mouse myeloma has been purified to near homogeneity, and its properties have been examined. The enzyme did not catalyze a detectable level of dNTP turnover, pyrophosphate exchange, pyrophosphorolysis, 3'-exonuclease degradation, or 5'-exonuclease degradation. Steady-state kinetic studies point to an ordered bibi mechanism for the polymerization reaction. Metal activation, which is required for polymerization, did not alter the Km for either the dNTP or the template--primer.

Animals↗
PubMed1979

DNA polymerase beta from brain neurons is a repair enzyme.

DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases.

Animals↗
PubMed1979

Functional roles of DNA polymerases beta and gamma.

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) beta and gamma were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase beta which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this way probably due to the inability of brain tissues to excise alkylated bases from DNA. The role of DNA polymerase gamma was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA replication occurs in mitochondria, and exploiting the fact that DNA polymerase gama is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase gamma in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase alpha, we conclude that DNA polymerase alpha is mainly responsible for DNA replication in nuclei, DNA polymerase beta is involved in nuclear DNA repair, and DNA polymerase gamma is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase alpha in DNA repair or for DNA polymerase beta in DNA replication cannot be excluded.

Animals↗
PubMed1976

Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.

Avian Leukosis Virus↗
PubMed1979

Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer.

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.

Animals↗
PubMed1979

Unique requirements for template primers of DNA polymerase beta from rat ascites hepatoma AH130 cells.

The optimal condition for the rat DNA polymerase beta activity with (rA)n . (dT)12-18 as a template-primer was determined. The activity was remarkably affected by the concentration of the primer, (dT)12-18' and the mixing ratio of (dT)12-18 to (rA)n. DNA polymerase beta requires higher primer concentration (Km = 11.1 microM with respect to 3'-OH of the primer) than DNA polymerase gamma (Km = 0.04 microM) or oncornaviral DNA polymerase (Km = 0.08 microM) and the enzyme represented the maximum activity in the base ratio of 2:1 with (dT)12-18 and (rA)n suggesting the difference in reaction mechanisms of these enzymes. Under the optimized conditions, the specific activity of the near homogeneous preparation of DNA polymerase beta was 1,000,000 units per mg protein.

Animals↗
PubMed1979

Difference in the mechanisms of poly(dT) synthesis by DNA polymerases beta and gamma.

Poly(dT) products which were synthesized depending on (rA)n . (dT)12-18 as a template . primer by mammalian DNA polymerases beta and gamma were analyzed by alkaline sucrose gradient centrifugation. The size of the population of poly(dT) chains synthesized by DNA polymerase beta increased slowly and consistently during incubation up to at least 30 min. On the other hand, the product size with DNA polymerase gamma reached the final size (7 s) within 5 min and the number of products increased during further incubation. Comparison of product number per enzyme molecule suggests that DNA polymerase beta acts on multiple primers in a distributive fashion while DNA polymerase gamma completes poly(dT) chains of large size in a one-by-one fashion.

DNA Polymerase II↗
PubMed1979

Additive effects of bleomycin and neocarzinostatin on degradation of DNA, inhibition of DNA polymerase beta, and cell growth.

The interactions of the two antitumor protein antibiotics, neocarzinostatin (NCS) and bleomycin (BLM), were studied on subcellular and cellular levels. BLM and NCS were found to remove thymine from double-stranded DNA. Combination experiments using BLM and NCS together in an assay with isolated DNA revealed an additive effect in splitting. Under limiting concentration conditions, BLM and NCS induce alkali-labile sites in DNA without a subsequent cleavage of the chain. After transfer of BLM- or NCS-treated DNA into an alkaline solution, strand scissions occur. Combination of BLM and NCS results in an additive DNA-cleaving effect, which indicates that the splitting reactions initiated by BLM or NCS are not influenced if the two antibiotics are applied in combination. The DNA polymerase beta is inhibited by BLM (at higher concentrations) and by NCS in a competitive way with respect to DNA. The inhibition constant of BLM and NCS in a combination experiment was found to be the result of the sum of the inhibition constants of BLM and NCS. Using L5178Y mouse lymphoma cells, it was found that cells incubated with both BLM and NCS show "unbalanced growth." The dose-response curves from BLM and NCS have identical slopes; they are characteristic for compounds which selectively inhibit DNA synthesis. By use of isobolograms, it could also be clearly shown that BLM and NCS interact additively.

Animals↗
PubMed1979

Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis.

The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis.

Animals↗
PubMed1979

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.

Arabinofuranosylcytosine Triphosphate↗
PubMed1979

Identification of a DNA polymerase beta-like form in Drosophila melanogaster adult flies.

DNA polymerase beta is widely distributed in the eukariotes. So far, few examples are known in which a DNA polymerase alpha -like form alone is reported. Surprisingly, DNA polymerase beta was not detected in Drosophila embryos, while it is present in the cells of multicellular species from sponge to mammals. In view of the relevance of Drosophila as a model biological system for studying the role of the various DNA metabolism enzymes in vivo we have reinvestigated the presence of the DNA polymerase beta-like form in Drosophila adult flies. Here we report the occurrence in Drosophila melanogaster adult flies of a DNA polymerase activity that, for its NEM(1) resistance, template specificity, sensitivity to ddTTP, sedimentation coefficient and nuclear localization can be classified as a beta-like form.

Animals↗
PubMed1977

Purification, biochemical characterization and serological analysis of cellular deoxyribonucleic acid polymerases and a reverse transcriptase from spleen of a patient with myelofibrotic syndrome.

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 template-primers 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 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.

Child, Preschool↗
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↗