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At least 19 recordsLinked to original sources
PubMed1977

Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria.

The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.

Animals↗
PubMed1977

Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei.

DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.

Animals↗
PubMed1979

Purification and properties of DNA polymerase gamma from rabbit intestinal epithelial cells.

1. DNA polymerase gamma from the cytoplasmic fraction of rabbit intestinal epithelial cells has been purified 120 000-fold and was free of phosphatase and nuclease activities towards deoxyribonucleoside-5'-triphosphates and polynucleotides. 2. The enzyme exhibited maximal activity for activated DNA and poly(A) . oligo(dT)12--18 at pH 8.5 IN 0.25 AND 0.15 M-KCl, respectively. Km values for dTTP with these two templates were 0.5 and 3.8 microM, respectively. 3. In contrast to DNA polymerases alpha and beta, the enzyme replicated poly(A) . oligo(dT)12--18 10 times faster and poly(dA) . oligo(dT)12--18 5 times slower than activated DNA. 4. DNA polymerase gamma did not replicate poly(C) . oligo(dG)12--18 or poly(Cm) . oligo(dT)12--18. The reaction with poly(I) and poly(U) did not exceed 1% of that observed with poly(A). 5. The enzyme was inhibited in 60% by antiserum against DNA polymerase gamma from human lymphoblasts. 6. The nuclear fraction of rabbit intestinal epithelial cells contained DNA polymerase gamma with the same characteristics.

Animals↗
PubMed1975

Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex.

The major DNA polymerase in a nuclear membrane complex that is capable of synthesizing viral DNA sequences in vitro has been purified about 900-fold from adenovirus 2-infected KB cells. The enzyme was characterized as belonging to the class of mammalian DNA polymerases (DNA polymerase gamma) that can utilize poly(A) with oligo(dT) as template primer.

Adenoviridae↗
PubMed1976

HeLa cell DNA polymerase gamma: further purification and properties of the enzyme.

DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.

DNA Polymerase I↗
PubMed1978

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.

Cell Line↗
PubMed1977

Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase gamma of human lymphoblasts.

An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.

Antigen-Antibody Reactions↗
PubMed1976

Deoxynucleotide-polymerizing enzyme activities in T- and B-cells of acute lymphoblastic leukemia origin.

All 5 thymus-dependent cell (T-cell) lines (Molt-3; Molt-4; RPMI-8402; CCRF-CEM; CCRF-HSB-2) and 7 thymus-independent cell (B-cell) lines (RPMI-8382, RPMI-8392, RPMI-8412, RPMI-8422, RPMI-8432, RPMI-8442, CCRF-SB) established so far from acute lymphoblastic leukemia patients were examined for deoxynucleotide polymerizing enzymes. All T- and B-cells had DNA polymerase gamma, DNA polymerase beta, and terminal deoxynucleotidyl transferase both in the soluble (the latter 2 enzymes only in small amounts) and chromatin fraction, whereas DNA polymerase alpha was found only in the soluble fraction. With respect to their sedimentation and chromatographic behavior, template-primer requirements, Km for deoxythymidine triphosphate or deoxyguanosine triphosphate divalent cation preference, effect of NaCI and inhibitors, the enzymes from T- and B-cells resembled each other and those from other mammalian cells. DNA polymerase alpha, beta, and gamma from T-cells like those from "fresh" acute lymphoblastic leukemia cells, were more thermolabile than those from B-cells or phytohemagglutinin-stimulated normal lymphocytes. In addition, the terminal deoxynucleotidyl transferase from the above cells was completely inactivated in 5 to 6 min at 50 degrees, whereas the DNA polymerase alpha, beta, and gamma retained considerable activity even after heating for 25 min at 50 degrees. DNA polymerase activity of the soluble fraction from T-cells was of the same magnitude as in B-cells when expressed on a DNA basis but twice that of B-cells when expressed on a protein basis. High terminal deoxynucleotidyl transferase activity, equivalent to that observed in acute lymphoblastic leukemia cells, was found in all T-cell lines that, when expressed on a DNA basis, was 30 to 100 times higher than the B-cell lines tested. These results support the suggestion of earlier investigators that T-cell lines examined here may have originated from leukemic cells.

B-Lymphocytes↗
PubMed1977

DNA polymerase gamma of rabbit intestinal epithelial cells.

1. DNA-directed DNA polymerase gamma was isolated from epithelial cells of the rabbit small intestine, and characterized. 2. The molecular weight of the enzyme, determined by Sephadex G-200 filtration was 105 000 +/- 15%. 3. The enzyme showed preference for poly(A) replication on poly(A)-poly(dT) or poly(A)-oligo(dT) 12-18 templates, as compared with activated DNA. Poly(C)-oligo(dG)12-18 replication was not observed. 4. The Michaelis constants for dTTP in replication of activated DNA and poly(A)-oligo(dT)12-18 were 3.3 and 2.0 micron, respectively.

Animals↗
PubMed1979

Recognition of structure of 5-halogenated derivatives of ara-UTP by DNA polymerase gamma and reverse transcriptase.

This report deals with the test of a series of 5-halogenated derivatives of ara-UTP for the inhibition of DNA polymerase gamma and viral reverse transcriptase. The compounds newly synthesized and tested were; ara5-FUTP, ara5-C1UTP, ara5-BrUTP and ara5-IUTP. The results were: 1) All these compounds were inhibitory to DNA polymerase gamma and reverse transcriptase. The mode of inhibitions was, in all cases, competitive against dTTP. 2) Ki values for these inhibitors with DNA polymerase gamma were lower than those with reverse transcriptase. 3) Ara5-ClUTP was less inhibitory to reverse transcriptase than other derivatives.

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↗
PubMed1978

Inhibition of deoxynucleotide-polymerizing enzyme activities of human leukemia lymphoblasts and simian sarcoma virus by tilorone and thirteen of its analogs.

Tilorone, which is 2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one dihydrochloride, and 13 of its analogs inhibited human cellular DNA polymerases alpha and beta assayed with activated DNA as template and also cellular DNA polymerase gamma and DNA polymerase from simian sarcoma virus assayed with poly(A) (dT)12-18 as template. Terminal deoxynucleotidyltransferase (TdT), which has no template requirement, was not inhibited by any of the 14 compounds when d(A)12-18 or d(G)12-18 was used as initiator. Three compounds did not inhibit TdT assayed with activated DNA as initiator, but 11 compounds did, and these 11 compounds were generally less inhibitory to TdT than to the other DNA polymerases. The three compounds that did not inhibit TdT assayed with activated DNA but did inhibit the other DNA polymerases will be useful in the characterization of TdT activity. Modifications of the polycyclic ring structure of tilorone and the kinds of substituent groups attached to the ring structures influenced the degree of inhibition of all enzymes.

DNA↗
PubMed1979

Identification of gamma-like DNA polymerase from sea urchin embryos.

A gamma-like DNA polymerase devoid of DNA polymerase-alpha and -beta activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) . oligo(dT)12--18 followed by poly(dA) . oligo(dT)12--18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-gamma.

Animals↗
PubMed1978

[DNA polymerases in human platelets].

Human platelets have two DNA polymerases. In the mitochondrial-free cytoplasm we have found a DNA polymerase gamma and another DNA polymerase very closely related to the polymerase alpha from animal cells. In the mitochondria only the gamma activity is present.

Blood Platelets↗
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↗