共查询到20条相似文献,搜索用时 15 毫秒
1.
R Takada-Takayama F Hanaoka M Yamada M Ui 《The Journal of biological chemistry》1991,266(24):15716-15718
We have purified DNA polymerase alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA polymerase alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA polymerase alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA polymerase alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant polymerase indicated that the temperature-sensitive mutation in DNA polymerase alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme. 相似文献
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DNA polymerase alpha 总被引:16,自引:0,他引:16
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A DNA polymerase alpha-primase complex, which had been purified by means of immunoaffinity column chromatography, showed little activity in a reaction mixture composed of Tris-HCl buffer, but showed full activity in potassium phosphate buffer. It was found that potassium ion is required for the reaction by the immunoaffinity-purified enzyme. On the other hand, the DNA polymerase alpha purified by the orthodox biochemical method showed full activity in both buffer systems. A protein factor, which could restore the activity of immunoaffinity-purified DNA polymerase alpha-primase complex in the potassium-free reaction mixture, was separated from biochemically purified DNA polymerase alpha. The factor, designated as factor T, was stable to heat up to 70 degrees C, but was sensitive to trypsin. It sedimented at about 4S through a glycerol gradient. SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands at 56 and 54 kDa. By immunoprecipitation, the factor T was shown to be physically associated with DNA polymerase alpha-primase complex. The stimulation was also observed with poly[d(A-T)], primed M13 DNA, and heat-denatured DNA. 相似文献
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Detection and characterization of a novel factor that stimulates DNA polymerase alpha 总被引:2,自引:0,他引:2
A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition. 相似文献
6.
V L Sylvia C O Joe J O Norman G M Curtin D L Busbee 《Biochemical and biophysical research communications》1986,135(3):880-885
DNA polymerase alpha was activated in vitro by cAMP-independent, phospholipid-dependent, protein kinase catalytic subunit. Of the phospholipids examined, phosphatidylinositol showed the greatest potential for interaction with protein kinase and ATP to activate DNA polymerase alpha in vitro. DNA polymerase alpha was directly activated by phosphorylated phosphatidylinositol in the absence of protein kinase and ATP. Activation of DNA polymerase alpha as a function of phosphorylation was demonstrated using 32P-ATP as the phosphate donor. In vitro treatment of the enzyme with phosphatidylinositol produced Linweaver-Burk plots showing noncompetitive kinetics of enzyme activation, suggesting that activation occurs prior to binding of the enzyme to DNA template/primer. These data indicate that DNA polymerase alpha may be activated in vitro in the presence of protein kinase, ATP, and phosphatidylinositol, and suggest that phosphorylation of the enzyme may constitute an intracellular mechanism of enzyme activation. 相似文献
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A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides. 相似文献
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A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast. 相似文献
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Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well. 相似文献
10.
Bleomycin is an important anti-tumor agent which works primarily through it's degradation of DNA template. Using synthetic single (poly[dA]-oligo-[dT]) and double stranded (poly[dA-dT]) templates, we noted significant inhibition when the BLM resistant homopolymer was used. Furthermore, when each of the components of the DNA polymerase assay were treated with bleomycin separately, followed by removal of bleomycin, significant inhibition (35%) of the enzyme was observed. The limited inhibition of DNA polymerase by BLM was attributed to residual activity of the enzyme-inhibitor complex. 相似文献
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P Zahradka 《FEBS letters》1987,212(2):259-262
DNA polymerase alpha was purified from human KB cells by immunoaffinity chromatography. Enzyme activity was inhibited by three different monoclonal antibodies (SJK-132, SJK-211, SJK-287). Kinetic analysis showed that each antibody neutralized polymerase activity by a different mechanism. SJK-132 was competitive with DNA indicating it interacts with the DNA binding domain of the polymerase. SJK-287 showed a biphasic response to dCTP suggesting two dCTP binding sites exist on polymerase alpha. SJK-211 was non-competitive with DNA, dCTP and dATP. 相似文献
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Steady-state kinetic studies of DNA polymerase alpha purified from mouse myeloma MOPC104E cells have been carried out. The results of initial velocity analysis with or without sodium pyrophosphate, a product inhibitor, indicated that the reaction mechanism of this enzyme can be categorized as an ordered Bi Bi type where the concentration of the ternary complex is very low. 相似文献
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Pavlov YI Frahm C Nick McElhinny SA Niimi A Suzuki M Kunkel TA 《Current biology : CB》2006,16(2):202-207
Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability. 相似文献
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Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties 总被引:3,自引:0,他引:3
S W Krauss D Mochly-Rosen D E Koshland S Linn 《The Journal of biological chemistry》1987,262(8):3432-3435
Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha. 相似文献
16.
O I Lavrik G A Nevinski? I A Potapova N B Tarusova O V Khalabuda 《Molekuliarnaia biologiia》1989,23(2):400-408
The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template. 相似文献
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The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms. 相似文献
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Mechanism of DNA polymerase alpha inhibition by aphidicolin 总被引:9,自引:0,他引:9
Synthetic oligonucleotides of defined sequence were used to examine the mechanism of calf thymus DNA polymerase alpha inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol alpha-DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol alpha.DNA.aphidicolin ternary complex, while DNase I protection experiments provide direct physical evidence. When deoxyguanosine is the next base to be replicated, Ki = 0.2 microM. In contrast, the Ki is 10-fold higher when the other dNMPs are at this position. Formation of a pol alpha.DNA.aphidicolin ternary complex did not inhibit the primase activity of the pol alpha.primase complex. Neither the rate of primer synthesis nor the size distribution of primers 2-10 nucleotides long was changed. Elongation of the primase-synthesized primers by pol alpha was inhibited both by ternary complex formation using exogenously added DNA and by aphidicolin alone. 相似文献
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Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication 总被引:17,自引:0,他引:17
DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献