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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.
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PMID:Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex. 42 7

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. 354 18

Poly(dC,3- MedC ) has been synthesised and used as a template to compare the miscoding properties of 3-methylcytosine (3-MeC) during DNA and RNA synthesis. Although 3-MeC was promutagenic with the RNA polymerase incorporating both AMP and UMP in the ratio of approximately 5:1 (agreeing with results reported by earlier workers) no non-complementary nucleotide incorporation was observed with DNA polymerase I. The results show that 3-MeC, which is a strong inhibitor of DNA synthesis, is only promutagenic with the less accurate RNA polymerase and that the reported differences in promutagenicity for this modified base with the two nucleotide polymerising enzymes arise from different specificities for the two enzymes.
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PMID:Differences in the promutagenic nature of 3-methylcytosine as revealed by DNA and RNA polymerising enzymes. 637 42

A new modified polydeoxynucleotide, a copolymer of nucleotides of 2'-deoxyadenosine and the very efficacious anti-herpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine was synthesized with E. coli DNA polymerase I enzyme. It is characterized by its physical (absorption and circular dichroism spectra, thermal transition, sedimentation analysis) and bioorganic (template activity, stability) properties. Compared to poly [d(A-T)], the modified polydeoxynucleotide had a lower thermal stability but exhibited higher stability against DNases and higher template activity for DNA synthesis. Template activity for RNA synthesis of this template was, however, poor and extent of AMP and UMP incorporation was limited as well.
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PMID:Modified polynucleotides. VI. Properties of a synthetic DNA containing the anti-herpes agent (E)-5-(2-bromovinyl)-2'-deoxyuridine. 675 97

The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release [33P]UMP when incubated with a RNA.DNA hybrid containing a [33P]UMP-labeled RNA strand. The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator. RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent. A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion. The radiolabeled RNA.DNA hybrid or DNA.DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times. The DNA.DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration. However, nearly complete digestion of the RNA.DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA.DNA hybrid vs the DNA.DNA duplex. Time course reactions of the enzymatic digestion of the 3'-labeled RNA.DNA hybrid or DNA.DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.
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PMID:Properties of the 5'-->3' exonuclease/ribonuclease H activity of Thermus thermophilus DNA polymerase. 771 Oct 21

A series of beta-alkylaminopropiophenone derivatives were demonstrated to be potent antineoplastic agents. Several compounds showed activity against Ehrlich ascites carcinoma growth in CF1 mice by demonstrating over 70% inhibition. Most of these agents proved to be potent cytotoxic agents in inhibiting the growth of a number of murine and human cancer cell lines grown in tissue culture. Their ED50 values were comparable to those of the selected standard anticancer drugs, such as 6-MP, ara-C, hydroxyurea, 5-FU, 6-aza-UMP, etoposide, antimycin A, actinomycin D and cycloheximide. In the mode of action studies in Tmolt3 cells, beta-(3",5"-dimethyl)piperidinopropiophenone was observed to reduce DNA and RNA synthesis significantly at 25 microM within 60 min incubation. The site of action of this agent appears to involve the reduction of the activities of Tmolt3 DNA polymerase alpha 1 dihydrofolate reductase, PRPP-amido transferase and ribonucleoside reductase.
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PMID:Antineoplastic activities of 2,3,4-chloro-substituted beta-alkylaminopropiophenone derivatives in CF1 mice and in murine and human tumor cells. 886 31

Terminal RNA uridylyltransferases (TUTases) catalyze the transfer of UMP residues to the 3' hydroxyl group of RNA. These enzymes belong to the DNA polymerase beta superfamily, which also includes poly(A) polymerases, CCA-adding enzymes, and other nucleotidyltransferases. Studies of uridylyl insertion/deletion RNA editing in mitochondria of trypanosomatids provided the first examples of biological functions for TUTases: posttranscriptional uridylylation of guide RNAs by RNA editing TUTase 1 (RET1) and U-insertion mRNA editing by RNA editing TUTase 2 (RET2). The editing TUTases are unified by the presence of conserved catalytic and nucleotide base recognition domains, yet differ substantially in auxiliary function-specific domains, quaternary structure, RNA substrate specificity, and processivity. This chapter describes isolation of TUTases and their complexes from trypanosomatids, methods used for analysis of interactions involving RET1 and RET2, purification of recombinant proteins, and enzyme kinetic assays.
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PMID:RNA editing uridylyltransferases of trypanosomatids. 1766 36

Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.
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PMID:Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases. 1778 18

RNA-specific nucleotidyl transferases (rNTrs) are a diverse family of template-independent polymerases that add ribonucleotides to the 3'-ends of RNA molecules. All rNTrs share a related active-site architecture first described for DNA polymerase beta and a catalytic mechanism conserved among DNA and RNA polymerases. The best known examples are the nuclear poly(A) polymerases involved in the 3'-end processing of eukaryotic messenger RNA precursors and the ubiquitous CCA-adding enzymes that complete the 3'-ends of tRNA molecules. In recent years, a growing number of new enzymes have been added to the list that now includes the "noncanonical" poly(A) polymerases involved in RNA quality control or in the readenylation of dormant messenger RNAs in the cytoplasm. Other members of the group are terminal uridylyl transferases adding single or multiple UMP residues in RNA-editing reactions or upon the maturation of small RNAs and poly(U) polymerases, the substrates of which are still not known. 2'-5'Oligo(A) synthetases differ from the other rNTrs by synthesizing oligonucleotides with 2'-5'-phosphodiester bonds de novo.
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PMID:RNA-specific ribonucleotidyl transferases. 1787 11