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1.
2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis. 相似文献
2.
The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed. 相似文献
3.
H Suzuki M Menegazzi A C De Prati T Ogura H Esumi A Matsukage M Libonati 《Biochemical and biophysical research communications》1991,181(2):623-628
On induction of proliferation of human peripheral blood mononuclear cells by phytohemagglutinin treatment, DNA polymerase beta activity increases markedly before and during DNA replication. The increase of enzymatic activity seems to be well correlated with the increase of DNA polymerase beta mRNA, which is induced by enhanced expression of the DNA polymerase beta gene. These data suggest that DNA polymerase beta is involved in DNA repair, which is linked to replicative DNA synthesis, or directly in replicative DNA synthesis in normal proliferating cells. 相似文献
4.
Unscheduled DNA synthesis was induced by bleomycin in isolated rat liver nuclei and in permeable mouse ascites sarcoma cells. ATP significantly enhanced the bleomycin effect of inducing unscheduled DNA synthesis. Replicative DNA synthesis in permeable mouse ascites sarcoma cells was inhibited by bleomycin. The apparent inhibition or stimulation by bleomycin of in vitro DNA synthesis was thought to be determined by a balance between inhibited DNA replicase activity and induced unscheduled DNA synthesis. 相似文献
5.
A comparative study of the protein components of ribonucleoprotein particles isolated from rat liver and hepatoma nuclei 总被引:2,自引:0,他引:2
To solve the mechanism for the complete cessation of DNA synthesis in Tetrahymena cells involved in the amino acid starvation, the nature of DNA polymerase activity was investigated in crude enzyme preparations or in toluene-permeabilized specimens. In crude enzyme preparations from growing cells, 3H-TTP incorporation into acid-insoluble products showed little dependency on exogenous DNA template, while incorporation increased markedly in the presence of ATP. These characteristics were very similar to those of replicative DNA synthesis in permeabilized Escherichia coli.Variations of DNA and RNA polymerase activities following transfer of exponentially growing Tetrahymena cells to amino acid-deprived medium showed that in the crude enzyme preparations DNA polymerase activity dropped sharply within 3 h after the transfer and practically no activity was detected thereafter, whereas RNA polymerase activity did not disappear in the same preparations. Such enzyme kinetics coincided well with the kinetics of in vivo synthesis of the corresponding nucleic acid.The cessation of DNA synthesis in the amino acid-starved cells may be due not to the activation of DNase or a soluble polymerase inhibitor, nor to the deficiency of each kind of deoxyribonucleoside triphosphate or magnesium ion or ATP generation system. It follows from this that the cessation of DNA polymerase activity in the starved cells may be due to the deficiency of DNA polymerase or its associated factor(s) as a reflection of short life-span of such a protein. 相似文献
6.
Biochemical aspects of cardiac muscle differentiation. Determination of deoxyribonucleic acid template availability and 3′-hydroxyl termini in nuclei and chromatin by using exogenous deoxyribonucleic acid polymerases
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William C. Claycomb 《The Biochemical journal》1978,171(2):289-298
Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase alpha) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3'-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase alpha are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase alpha under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3'-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3'-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3'-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases. 相似文献
7.
8.
Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of alpha-type polymerases (resolved into alpha1, 8 S, and alpha 2, 6 S, in the present experiments), while the infection little affected the level of beta-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in alpha-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase alpha, but not of the polymerases alpha2 and beta. These results support the idea that alpha-type polymerases are involved in the replicative synthesis of cellular and viral DNA. 相似文献
9.
The paper deals with the effect of the single-strand (ss) DNA-binding proteins (SSB-proteins) from the Ehrlich ascites tumor (EAT) cells and from the eggs of silkworm, as well as the mouse serum blood proteins, having preferential affinity to ss DNA, on the DNA replicative synthesis in the EAT cells permeable for the macromolecules, and, for the silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm permeable for macromolecules. SSB-proteins of EAT to considerable extent stimulated the DNA synthesis. At the same time, the other proteins (from the silkworm and from the serum) activated the DNA synthesis in the permeable cells to the less extent. It was found that SSB-proteins from the silkworm had a 1.5-13 fold stimulating effect on the DNA replicative synthesis in the homologous system (in the permeable nuclei). If the permeability for the macromolecules of the cells and nuclei treatment with Triton X-100 may be different, it is supposed that the activation of the DNA synthesis by the exogenous proteins depends on the homologous system of the DNA replicative complex. It is possible that the effect of the serum proteins on the DNA synthesis is connected with the masking of the ss regions of DNA which inhibited DNA-polymerase alpha. Perhaps the mechanisms of the activation of the DNA replicative synthesis by the proteins in vitro with the purified DNA polymerase alpha and in vivo are of different nature and are conditioned by homology of the deoxyribonucleoproteins. 相似文献
10.
The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents 总被引:11,自引:0,他引:11
The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed. 相似文献
11.
An RNAase-sensitive DNA polymerase from rat cells transformed by avian sarcoma virus has been characterized. The enzyme requires RNA for its activity, as shown by its sensitivity to RNAase with endogenous as well as exogenous DNA templates. This sensitivity is maintained after its purification by sucrose gradients and ion exchange columns. A molecular weight of about 100 000 has been estimated. This DNA polymerase requires high salt concentration for its activity, is resistant to high concentrations of phosphonoacetic acid (400 micrograms/ml), is partially inhibited by 5 mM N-ethylmaleimide, and is completely inhibited by 0.3 mM parahydroxymercuribenzoate. 相似文献
12.
Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase 总被引:6,自引:0,他引:6
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Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase. J. Bacteriol. 91:2327-2332. 1966.-Mengovirus induces the appearance of a ribonucleic acid (RNA) polymerase activity in Novikoff hepatoma cells which is readily distinguished from the deoxyribonucleic acid (DNA)-dependent RNA polymerase since it is not inhibited by actinomycin D or deoxyribonuclease, but is inhibited by ammonium sulfate, and is stable at -17 C. The incorporation of uridine into RNA by infected cells in the presence of actinomycin D does not reflect the viral polymerase activity as measured in cell-free preparations. The viral-induced RNA polymerase is produced in a biphasic fashion. Puromycin inhibits the production of viral polymerase, and in its presence the enzyme appears to be unstable between 4 and 6 hr. Puromycin also prevents the secondary rise in polymerase which begins at the end of replicative cycle. Under these conditions, however, the polymerase appears to be stable. The overall data indicated that some unspecified process is responsible for the apparent instability of viral-induced RNA polymerase between 4 and 6 hr and that it becomes inoperative toward the end of the replicative cycle. 相似文献
13.
A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication. 总被引:1,自引:1,他引:0
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H Pospiech I Kursula W Abdel-Aziz L Malkas L Uitto M Kastelli M Vihinen-Ranta S Eskelinen J E Syvoja 《Nucleic acids research》1999,27(19):3799-3804
The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication. 相似文献
14.
Preferential Inhibition of Herpes-Group Viruses by Phosphonoacetic Acid: Effect on Virus DNA Synthesis and Virus-Induced DNA Polymerase Activity 总被引:4,自引:0,他引:4
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Eng-Shang Huang Chien-Hui Huang Shu-Mei Huong Maryjane Selgrade 《The Yale journal of biology and medicine》1976,49(1):93-98
In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent. 相似文献
15.
Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells 总被引:8,自引:0,他引:8
Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently. 相似文献
16.
17.
Mary Jane Vrooman Marjorie H. Barnes Neal C. Brown 《Molecular & general genetics : MGG》1978,164(3):335-339
Summary The characteristics of Bacillus subtilis dnaF, a mutation specifying a temperature sensitive phenotype, were examined to determine its relationship to polC, the gene specifying the structure of DNA polymerase III (pol III). Exposure of growing cells bearing dnaF to non-permissive temperature inhibited replicative DNA synthesis and specifically depressed the expression of pol III activity in crude extracts. Highly purified pol III derived from cells bearing dnaF was temperature sensitive in its polymerase activity, indicating that dnaF is a specific, polC mutation which specifies a structurally altered enzyme. 相似文献
18.
Nedelcheva MN Roguev A Dolapchiev LB Shevchenko A Taskov HB Shevchenko A Stewart AF Stoynov SS 《Journal of molecular biology》2005,347(3):509-521
The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled. 相似文献
19.
Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro. 相似文献
20.
Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis. 总被引:3,自引:1,他引:2
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The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP. 相似文献