共查询到20条相似文献,搜索用时 0 毫秒
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Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases. 相似文献
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Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA 总被引:1,自引:0,他引:1
J Sági E De Clercq A Szemz? A Csárnyi T Kovács L Otv?s 《Biochemical and biophysical research communications》1987,147(3):1105-1112
The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT). 相似文献
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V N Podust T O Korobe?nicheva G A Nevinski? V A Rikhter T I Abramova O I Lavrik 《Bioorganicheskaia khimiia》1990,16(2):226-235
Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase. 相似文献
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K Ono M Ogasawara A Matsukage 《Biochemical and biophysical research communications》1979,88(4):1255-1262
2′,3′-Dideoxythymidine 5′-triphosphate was found to strongly inhibit the activity of DNA polymerase α from mouse myeloma in the presence of manganese ion as divalent cation. The extent of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate increased by raising pH of the reaction. The mode of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate was competitive to the substrate, 2′-deoxythymidine 5′-triphosphate. Ki of the DNA polymerase α for 2′,3′-dideoxythymidine 5′-triphosphate (0.035 μM) was much lower than Km for 2′-deoxythymidine 5′-triphosphate (1.8 μM). 相似文献
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Time course of incorporation and the effect of 5'-triphosphate of the selective antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (bv5dUTP) on the incorporation of dTTP and dATP into template-primers of different structure were studied in E. coli DNA polymerase I Klenow fragment enzyme-catalyzed reactions. bv5dUTP could substitute for dTTP depending on the structure of template-primer. E.g. into calf thymus DNA incorporation of bv5dUMP was around 80% of that of dTMP at 30 minutes of incubation. The analog has also inhibited dTMP incorporation, net DNA synthesis, however, was hardly affected. The substrate properties of the analog were studied with [2-14C]-labelled bv5dUTP. 相似文献
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3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation. 相似文献
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The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage. 相似文献
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