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)

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.
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PMID:In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61. 789 Jun 5

Fluorescence depolarization decays were measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer.27-mer oligonucleotides bound to Klenow fragment of DNA polymerase I. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with two footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3- to 4-fold increase in the equilibrium partitioning of DNA into the exonuclease site; the largest effects were observed for purine-purine mismatches. Two or more consecutive G.G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250-fold greater than that of the corresponding matched DNA sequence. Internal single mismatches produced larger effects than the same mismatch at the primer terminus, with a delta delta G relative to the matched sequence of -1.1 to -1.3 kcal/mol for mismatches located 2, 3, or 4 bases from the primer terminus. Although part of the observed effects may be attributed to the increased melting capacity of the DNA, it appears that the polymerase site also promotes movement of DNA into the exonuclease site by rejecting aberrant primer termini. These effects suggest that the polymerase and exonuclease sites act together to recognize specific errors that distort the primer terminus, such as frameshifts, in addition to proofreading misincorporated bases.
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PMID:Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I. 793 11

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

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
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PMID:Studies of gapped DNA substrate binding by mammalian DNA polymerase beta. Dependence on 5'-phosphate group. 802 71

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.
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PMID:Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development. 808 46

In the present work, we have studied in vitro replication of N-2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II (cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the alpha subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G1G2CG3CC-) have been used in primer extension assays. Site-specifically platinated 5'd(ApG) or 5'd(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the NarI site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3'-->5' proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf (exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.
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PMID:Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerases. 810

Triple helices can be formed on single-stranded oligopurine target sequences by composite oligonucleotides consisting of two oligonucleotides covalently linked by either a hexaethylene glycol linker or an oligonucleotide sequence. The first oligomer forms Watson-Crick base pairs with the target, while the second oligomer engages in Hoogsteen base pairing, thereby acting as a molecular clamp. The triple-helical complex formed by such an oligonucleotide clamp, or "oligonucleotide-loop-oligonucleotide" (OLO), is more stable than either the corresponding trimolecular triple helix or the double helix formed upon binding of the oligopyrimidine complement to the same oligopurine target. Attaching a psoralen derivative to the 5' end of the OLO allowed us to photoinduce a covalent linkage to the target sequence. The psoralen moiety became covalently linked to all three portions of the triplex, thereby making the oligonucleotide clamp irreversible. These crosslinking reactions introduced strong stop signals during DNA replication, as shown on a plasmid containing a portion of the HIV proviral sequence of human immunodeficiency virus. A 16-mer oligopurine sequence corresponding to the "polypurine tract" of human immunodeficiency virus was chosen as a target for a psoralen-OLO conjugate. Three different stop signals for DNA polymerase were observed, corresponding to different sites of polymerase arrest on its template. Even in the absence of photoinduced crosslinking, the psoralen-OLO conjugate was able to arrest DNA replication. The formation of triple-helical structures on single-stranded targets may provide an alternative to the antisense strategy for the control of gene expression.
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PMID:Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target. 823 49

Elucidation of the structure of DNA by Watson and Crick [Nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on DNA replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [Watson, Science 248 (1990) 44-49]. I am totally convinced of the great importance of the Human Genome Project, and toward achieving this goal I strongly favor 'top-down' approaches consisting of the physical mapping and preparation of contiguous 50-100-kb fragments directly from the genome, followed by their automated sequencing based on the rapid assembly of primers by hexamer ligation together with primer walking. Our 'top-down' procedures totally avoids conventional cloning, subcloning and random sequencing, which are the elements of the present 'bottom-up' procedures. Fragments of 50-100 kb are prepared in sufficient quantities either by in vitro excision with rare-cutting restriction systems (including Achilles' heel cleavage [AC] or the RecA-AC procedures of Koob et al. [Nucleic Acids Res. 20 (1992) 5831-5836]) or by in vivo excision and amplification using the yeast FRT/Flp system or the phage lambda att/Int system. Such fragments, when derived directly from the Escherichia coli genome, are arranged in consecutive order, so that 50 specially constructed strains of E. coli would supply 50 end-to-end arranged approx. 100-kb fragments, which will cover the entire approx. 5-Mb E. coli genome. For the 150-Mb Drosophila melanogaster genome, 1500 of such consecutive 100-kb fragments (supplied by 1500 strains) are required to cover the entire genome. The fragments will be sequenced by the SPEL-6 method involving hexamer ligation [Szybalski, Gene 90 (1990) 177-178; Fresenius J. Anal. Chem. 4 (1992) 343] and primer walking. The 18-mer primers are synthesized in only a few minutes from three contiguous hexamers annealed to the DNA strand to be sequenced when using an over 100-fold excess of hexamers and T4 DNA ligase at room temperature, preferably in the presence of the single-strand-binding (SSB) protein of E. coli. These 18-nt primers are immediately extended by the DNA polymerase, Sequenase 2.0, in the dideoxy sequencing reaction. Very high quality sequencing ladders are obtained for single-stranded DNA or denatured double-stranded approx. 50-kb fragments, as exemplified by phage lambda DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:From the double-helix to novel approaches to the sequencing of large genomes. 827 70

We found that 28-mer phosphorothioate oligodeoxynucleotides (S-oligos) with and without sequence specificity complementary to Epstein-Barr virus (EBV) genes are potent inhibitors of EBV replication in cell culture. The decrease in the amount of EBV DNA, the activity of intracellular viral DNA polymerase, and virus production were dose dependent, with a 90% inhibitory dose of approximately 0.5 microM. No inhibition of cell growth was observed with the S-oligos at concentrations up to 20 microM. The mechanism of action appears to be the inhibition of EBV DNA synthesis. The reversibility of anti-EBV action is dependent on the dose and duration of drug exposure. S-oligos should be considered a new class of anti-EBV agents.
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PMID:Potent inhibition of Epstein-Barr virus by phosphorothioate oligodeoxynucleotides without sequence specification. 839 89


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