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)

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.
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PMID:(E)-5-(2-bromovinyl)-2'-deoxyuridine-5'-triphosphate as a DNA polymerase substrate. 733 97

DNA polymerase (EC 2.7.7.7) beta was purified to near homogeneity from chick embryos. The final preparation has a specific enzyme activity of 1,100,000 units (nanomoles of dTMP incorporation/h) per mg of protein with (rA)n X (dT)12-18 as a template-primer. The molecular weight of DNA polymerase beta is about 40,000 as judged by gel filtration on Sephadex G-150 column. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the polypeptide of Mr = 40,000 accounted for 95% of total protein in the final preparation. The fingerprint analysis of tryptic peptides of polypeptide shows that 14 out of 24 125I-peptides from the Mr = 40,000 polypeptide are the same as those of the Mr = 40,000 polypeptide of DNA polymerase beta purified from rat ascites hepatoma cells. A Mr = 27,000 polypeptide, which was present in the less pure preparation, did not show any structure similar to Mr = 40,000 polypeptides from rat and chick cells. Thus, it is concluded that chick embryo DNA polymerase beta consists of a single polypeptide of Mr = 40,000. The minimal number of DNA polymerase beta molecules per chick embryo cell was estimated as about 5,000.
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PMID:Chick embryo DNA polymerase beta. Purified enzyme consists of a single Mr = 40,000 polypeptide. 743 Jan 8

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
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PMID:Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine. 752 86

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.
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PMID:8-Hydroxyadenine (7,8-dihydro-8-oxoadenine) induces misincorporation in in vitro DNA synthesis and mutations in NIH 3T3 cells. 765 12

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.
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PMID:Metabolism and mode of selective inhibition of human immunodeficiency virus replication by 3'-azido-2',3'-dideoxy-5-iodouridine and 3'-azido-2',3'-dideoxy-5-bromouridine. 767 40

3,N4-Etheno-2'-deoxycytidine, 3-(hydroxyethyl)-2'-deoxyuridine, and 3,N4-ethano-2'-deoxy-cytidine are found in DNA of cells treated with either vinyl chloride or 1,3-bis(2-chloroethyl)-nitrosourea. These exocyclic and related DNA adducts were incorporated into oligodeoxynucleotides, which were then used as templates for primer extension in reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The miscoding potential of each lesion was determined quantitatively. DNA primers were readily extended on an epsilon dC-modified template; dAMP and dTMP were incorporated opposite the lesion. With high concentrations of DNA polymerase, small amounts of fully extended reaction products containing dAMP and dGMP or one-base and two-base deletions opposite ethano-dC were formed. Primer extension was blocked partially on templates containing 3-(hydroxyethyl)-dU; dAMP and smaller amounts of dTMP and dCMP were incorporated. The frequencies of nucleotide insertion opposite each of the three lesions and the frequencies of chain extension from the 3'-primer terminus, determined by kinetic analysis, were consistent with results of experiments utilizing polyacrylamide gel electrophoresis. We conclude from these studies that epsilon dC, ethano-dC, and 3-(hydroxyethyl)-U are potentially miscoding lesions; only epsilon dC facilitates translesional synthesis.
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PMID:Miscoding by the exocyclic and related DNA adducts 3,N4-etheno-2'-deoxycytidine, 3,N4-ethano-2'-deoxycytidine, and 3-(2-hydroxyethyl)-2'-deoxyuridine. 770 60

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.
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PMID:Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine. 770 90

Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incorporation of a nucleotide opposite the adduct and translesional synthesis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly across from the four different purine adducts we found that the deoxyadenosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG-AAII) allowed preferential incorporation of dCMP. Molecular dynamic simulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.
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PMID:Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid. 795 74

The alpha-anomer of deoxyadenosine (alpha-dA) is a major adenine lesion produced by hydroxyl radicals in DNA. To assess its biochemical effects on DNA replication, alpha-dA was site-specifically incorporated into oligodeoxyribonucleotide templates using phosphoramidite chemistry. alpha-dA in the template constituted a transient block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (polI), but translesional synthesis occurred after prolonged incubation. Primer extension assays and Maxam-Gilbert sequencing of newly synthesized products revealed that alpha-dA directed not only incorporation of the correct nucleotide, dTMP, opposite the lesion but also misincorporation of dAMP and dCMP. dGMP was barely incorporated under these conditions. The order of the incorporation frequency at the alpha-dA site was affected by the nearest neighbor base pair 3' to the lesion. T7 and Taq DNA polymerases, as well as RAV-2 reverse transcriptase, showed a selectivity similar to that of PolI with respect to the nucleotide incorporation opposite alpha-dA, suggesting that the discrimination of nucleotides associated with alpha-dA is independent of the origin of DNA polymerases and is an intrinsic feature of the lesion. The mutational spectrum predicted for alpha-dA (i.e., A-->G transitions and A-->T transversions) is significantly different from those reported for other hydroxyl radical induced DNA lesions such as abasic sites or 7,8-dihydro-8-oxoguanine, both primarily directing misincorporation of A. Possible biological consequences and the mechanism of dNTP discrimination associated with alpha-dA are discussed.
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PMID:Replication of DNA templates containing the alpha-anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals. 800 79


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