Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase. Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2).
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PMID:[Construction of a cDNA library of Schistosoma japonicum]. 171 36

3'-Mercapto-3'-deoxy-TTP was synthesized and tested as DNA chain terminator nucleotide for calf thymus alpha DNA polymerase, E. coli DNA polymerase I (Klenow fragment), terminal deoxyribonucleotidyltransferase (Bollum enzyme) and reverse transcriptase from AMV- and HIV-I-viruses. It was shown that the compound terminates DNA chain elongation by reverse transcriptases selectively and irreversibly. Other tested DNA polymerases do not use this nucleotide analogue as a substrate. 3'-Mercapto-3'-deoxythymidine was tested on lymphoblastoid T-cell line MT-4 with HIV-viruses and shown to suppress viruses as efficiently as 3'-azido-3'-deoxythymidine.
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PMID:[3'-Mercapto-3'-deoxythymidine-5'-triphosphate as a terminator of DNA synthesis catalyzed by RNA-dependent DNA-polymerases]. 171 4

A novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described. The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases. The modified nucleotide 9-(2-deoxy-5-O-triphospho-beta-D-ribofuranosyl)-N6,N6-ethano -2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2'-deoxyguanosine 5'-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase beta. Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage. The duplexes produced were found to be recognized as substrates by various DNA polymerases. The Km for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E. coli DNA polymerase I was found to be 29 microM, and the kcat was found to be 0.014 s-1. The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.
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PMID:A novel combined chemical-enzymatic synthesis of cross-linked DNA using a nucleoside triphosphate analogue. 198 67

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp221-Lys231 (peptide B8) and Cys234-Lys249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an alpha-helical array of 39 A which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase beta that has been implicated in the binding of DNA template.
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PMID:Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain. 200 41

We report here spectroscopic and biochemical data of a novel series of sugar-modified oligodeoxy-nucleotides, the carbocyclic oligothymidylates, c(dT)3-20. In c(dT)n a methylene group has been substituted for the oxygen atom of the deoxyribose ring of the natural thymidylate unit. c(dT)10-20 form helical structures, in contrast with oligothymidylates or poly(dT), based on absorbance versus temperature melting profiles. Secondary structure of c(dT)n, where n greater than 10 is assumed to be double helix. In addition to this, c(dT)n forms as a stable duplex with complementary poly(dA) as does parent (dT)n. On the other hand, c(dT)n-containing oligo/poly duplex is nearly inactive either as a template or as a primer in various DNA polymerase systems, and c(dT)n inhibits DNA replication as well. c(dT)n can efficiently be extended by terminal transferase and shows an increased nuclease stability compared to (dT)n. Base-pairing ability and nuclease stability of c(dT)n suggest that (+)-carbocyclic nucleoside-containing oligomers could be new potential antisense oligodeoxynucleotides.
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PMID:Biochemical properties of oligo [(+)-carbocyclic-thymidylates] and their complexes. 215 40

Four flavonoids (i.e., baicalein, quercetin, quercetagetin, and myricetin), known to be inhibitors of HIV-reverse transcriptase, have been shown to be more or less inhibitory to the activities of various cellular DNA and RNA polymerases. The degree of the inhibition varied depending on the combination of the flavonoid and the enzyme species: baicalein was moderately inhibitory to DNA polymerase gamma and E. coli DNA polymerase I; quercetin was strongly inhibitory to DNA polymerase beta and E. coli RNA polymerase and moderately inhibitory to DNA polymerase I; quercetagetin was a potent inhibitor for all of DNA polymerases alpha, beta, gamma, and I and RNA polymerase; myricetin was a strong inhibitor of DNA polymerases alpha and I and RNA polymerase. However, terminal deoxynucleotidyltransferase was virtually insensitive to inhibition by these flavonoids. The inhibition by the flavonoids was due to competition with the template.primer in the case of the DNA polymerases, whereas the inhibition was due to competition with the triphosphate substrate (GTP) in the case of RNA polymerase. The Ki values of these flavonoid inhibitors for DNA and RNA polymerases was determined.
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PMID:Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids. 229 90

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.
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PMID:Differential inhibition of various mammalian DNA polymerase activities by ammonium 21-tungsto-9-antimoniate (HPA23). 245 59

N7-Ethyldeoxyguanosine 5'-triphosphate (N7-Etd-GTP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at approximately 5% yield. N7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage. At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of greater than 48 h. An enzymatic method for placing an N7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3' to 5' exonuclease and 5' to 3' polymerase of the Klenow fragment of Escherichia coli DNA polymerase I. The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine, N6-methyladenine, and others).
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PMID:Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide. 266 93

A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.
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PMID:Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate. 271 38

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.
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PMID:Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region. 283 41


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