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)

Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites. Use of single-strand break frequencies to measure the number of repair events can therefore be in error by as much as a factor of 3. The blocked patches with free 3'OH termini were, on average, 22% of normal length, corresponding to between 6 and 17 bases (assuming a normal patch of 25-75 bases in length). Patches that remained unsealed in vivo were also resistant to sealing by T4 ligase in vitro. The data are more consistent with a mechanism of repair in which long single-strand gaps are first made by excision enzymes and subsequently filled in by DNA polymerase alpha. Strand displacement or nick translation mechanisms seem unlikely.
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PMID:Structure of repaired sites in human DNA synthesized in the presence of inhibitors of DNA polymerases alpha and beta in human fibroblasts. 640 37

Arabinocytidine and aphidicolin are inhibitors of alpha-DNA polymerase that have been shown to affect both normal DNA replication and repair synthesis in mammalian cells. In contradiction to the prevalent hypothesis that these inhibitors merely slow the polymerization rate at incision sites near lesions, our results suggest that the repair synthesis resistant to inhibitors is mediated by a separate pathway. Repair synthesis in contact-inhibited human cells following UV irradiation was inhibited 75-80% by arabinocytidine or aphidicolin, and most of the repair patches were not ligated into parental DNA, as judged by an enzymatic assay. However, the patches were not demonstrably shorter than those in untreated cells. Even following low-UV doses at which no inhibition of repair synthesis by the inhibitors was observed, a majority of the patches were not ligated. DNA polymerase beta is implicated in this alternate pathway, both by the known specificity of the inhibitors and by evidence from their sensitivity to S1 nuclease that the patches arise from displacement synthesis. The unligated patches are not degraded in vivo and eventually become ligated into parental DNA, very slowly in the presence of inhibitors but much more rapidly following their removal. Thus, under conditions of alpha-polymerase inhibition, a limited number of normal length repair patches are made, apparently by displacement synthesis, leaving displaced strands that remain substantially undegraded.
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PMID:Nature of DNA repair synthesis resistant to inhibitors of polymerase alpha in human cells. 642 5

The copy-DNA was synthesized on the mRNA fraction which was isolated from MOPC 21 mice myeloma by reverse transcription. This copy was completed with another chain without adding the exogenous primer, with the aid of the Klenov fragment of DNA polymerase I. After treatment of the double-stranded pin DNA with endonuclease S1, the poly(dA) sequences were built up using terminal deoxynucleotidyl transferase. The building up of the poly(dT) sequences at the DNA 3'-termini of pBR322 plasmid previously treated with BamHI was performed in a similar way. The average length of connecting polynucleotide sequences was 50 nucleotides. After annealing and transformation of the cells with Escherichia coli hybrid plasmids, selection of clones was made for ApRTcS phenotype. Further, we applied a stepwise selection of clones by means of increasing the specificity: colony hybridization, quantitative hybridization of the excess of plasmid DNA with (32P)-cDNA and hybridization of plasmid DNAs with mRNA units which were transferred from electrophoregrams to the diazo-papers. As a result, bacterial clones were selected which contained gene fragments showing the ability to hybridize with the RNA fraction characterized by mRNA mobility of the light-chain immunoglobulin G.
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PMID:[Construction and selection of bacterial clones hybridized with the mRNA of a light-chain immunoglobulin]. 680 31

Purified mRNA coding for chicken vitellogenin, a precursor of egg yolk proteins, was transcribed to complementary DNA (cDNAvit) with avian myeloblastosis virus (AMV) reverse transcriptase. Double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I (fragment A) using the self priming ability of the cDNA. Following S1 nuclease digestion the double-stranded cDNA was inserted into the Hind III site of plasmid pBR322 using the poly(dA) . poly(dT) tailing method, and the hybrid molecules were used to transform Escherichia coli chi 1776. Ampicillin-resistant colonies were screened by colony hybridization with 125I-labeled vitellogenen mRNA. Further screening of positive clones was done by agarose gel electrophoresis and in situ hybridization with 125I-labeled vitellogenin mRNA. In addition, plasmid DNA covalently bound to diazotized paper was used to select complementary mRNA sequences. The cloned vitellogenin sequences were shown to hybridize to a mRNA which directs the synthesis of immunoprecipitable vitellogenin when translated in a reticulocyte lysate cell-free system. The length of the inserted cDNA was determined by agarose gel electrophoresis and heteroduplex mapping. The largest insertion was about 2500 base pairs. Restriction mapping indicates that at least three plasmids out of four have different sequences.
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PMID:Recombinant plasmids containing avian vitellogenin structural gene sequences derived from complementary DNA. 698 72

The structure of chromatin is changed at early stages of glucocorticoid hormone interaction with rat hepatocytes. These changes consist in: a) increase of actidine orange binding in rate liver nuclei after injection of the hormone; b) decrease of the number of sites in chromatin which are sensitive to nuclease S1; c) inhibition of the nuclei capacity for DNA synthesis in the presence of E. coli DNA polymerase and d) increase of molecular weight of DNA fragments. The data obtained suggest that at early stages of hormonal induction part of template DNA ruptures is reconstituted, which can result in an increase of DNA molecular weights and changes in chromatin superstructure and the template properties of the protein.
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PMID:[Early changes in template DNA structure following glucocorticoid injection]. 711 5

This paper describes the enzyme-catalyzed in vitro synthesis of double-stranded DNA (ds-DNA) containing [3H]-labeled O(6)-ethylguanine (O(6)-EtGua), an alkylation product strongly implicated in mutagenesis and carcinogenesis by ethylating N-nitroso carcinogens. Single-stranded DNA (ss-DNA) containing O(6)ethyl-[8-3H]-2'-deoxyguanosine was synthesized using terminal deoxynucleotidyl transferase. The parameters determining yield of reaction, base ratios, and DNA chain length, were investigated. The O(6)-EtGua-containing ss-DNA could be replicated by DNA polymerase I, as indicated by the incorporation of [8,5'-3H]-2'-deoxyguanosine-5'-monophosphate and by the resistance of the replication product to nuclease S1 digestion. ds-DNA's with chain lengths between approximately 200 and 1000 base-pairs and O(6)-EtGua/guanine molar ratios of approximately 10(-2)--10(-3) were synthesized. Their use in the analysis of enzymatic mechanisms involved in the elimination of O(6)-alkylguanine from the DNA of mammalian cells is discussed. The procedure of synthesis described for (O(6)-EtGua)-containing ds-DNA may also be applicable for the production of ds-DNA containing structurally modified bases other than O(6)-EtGua.
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PMID:Enzymatic synthesis of double-stranded DNA containing radioactively labeled O(6)-ethylguanine as the only modified base. 727 13

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.
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PMID:Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli. 751 86

The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755-12763]. To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3. S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOL beta). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them.
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PMID:The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter. 759 Mar 51

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.
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PMID:Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection. 823 Apr 85

The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.
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PMID:Peptide nucleic acids (PNAs): potential antisense and anti-gene agents. 847 2


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