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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)

We have used an oligodeoxynucleotide of defined sequence to detect and quantitate proenkephalin mRNA in the poly(A)-containing fraction of RNA from bovine adrenal medullas. The decahexamer 5'-d(G-G-T-A-G-T-C-C-A-T-C-C-A-C-C-A)-3' was synthesized to be complementary to the codons specifying the amino acid sequence NH2-Trp-Trp-Met-Asp-Tyr-Gln-COOH. This stretch of amino acids occurs in peptide I, one of the intermediates in the biosynthetic pathway of the enkephalins in bovine adrenal medulla. This pathway starts with a precursor (proenkephalin) of about 45 kilodaltons [Stern, A. S., Jones, B. N., Shively, J. E., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 1962-1966]. The decahexamer hybridized to adrenal poly(A)+RNA and was extended into cDNA with reverse transcriptase (RNA-dependent DNA nucleotidyltransferase). Five main discrete products ranging in size from 115 to 168 nucleotides were observed. The sequences of these extensions were found to be identical over the approximately 70 nucleotides sequenced from their 5' termini and corresponded exactly to the sequence expected from the amino acid sequence of peptide I. These cDNAs and the decahexamer itself hybridized to an adrenal medullary poly(A)+RNA species of about 1500 nucleotides, sufficient in size to code for the proposed proenkephalin. At saturation, approximately 2 fmol of the decahexamer were bound per microgram of mRNA; thus, the proenkephalin mRNA represents about 0.1% of the total poly(A)+RNA population in the tissue.
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PMID:Detection and partial characterization of proenkephalin mRNA. 694 86

During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.
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PMID:On the fidelity of DNA replication. Nucleoside monophosphate generation during polymerization. 701 47

T4 DNA polymerase copolymerizes the SP isomers of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and 5'-O-(2-thiotriphosphate) with dTTP onto a poly(d(A-T) template in the presence of various metal ions. The corresponding RP diastereomers are inactive, independent of the metal ion used. The polymer resulting from the polymerization of the SP diastereomer of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and dTTP can be degraded by the 5' leads to 3' exonuclease activity of Escherichia coli DNA polymerase I and alkaline phosphatase (Brody, R. S., and Frey, P. A. (1981) Biochemistry 20, 1245-1252) to d(Tp(S)A). This material has the RP configuration as determined by comparison with the RP and SP diastereomers obtained by chemical synthesis and preparative separation by high performance liquid chromatography. This result indicates inversion of configuration at the alpha-phosphorus in the nucleotidyl transfer reaction and is compatible with the absence of a covalent enzyme intermediate.
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PMID:A study of the mechanism of T4 DNA polymerase with diastereomeric phosphorothioate analogues of deoxyadenosine triphosphate. 704 12

The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly [d(A-T)] or poly [d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases. The ability of Escherichia coli DNA polymerase I to copy over apurinic sites was demonstrated using single-stranded circular DNA of bacteriophage 0X174 as a template and starting DNA synthesis at a fixed point. Analysis of the newly synthesized 0X174 restriction fragments on neutral and alkaline sucrose gradients shows that synthesis proceeded past apurinic sites. When using depurinated 0X174 DNA containing the am3 amber mutation as a template for copying by E. coli DNA polymerase I, an increased reversion to wild type is observed after transfection into E. coli spheroplasts. The enhancement in reversion frequency is proportional to the extent of depurination, suggesting that depurination is also mutagenic during copying natural DNA in vitro. When noncopied depurinated 0X174 am3 DNA is transfected in E. coli spheroplasts, no increase in reversion frequency is observed above background level. However, when the spheroplasts are derived from bacteria in which the SOS response had been induced by UV irradiation, a substantial increase is observed for depurinated molecules, whereas no increase is observed for nondepurinated templates, suggesting in vivo mutagenesis at depurinated sites. In each of the different assay systems investigated, the increase in misincorporation or reversion frequency is a linear function of the number of sites and is abolished by treatment of the depurinated templates with alkali, which rapidly induces strand breakage at apurinic sites.
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PMID:Depurination of DNA as a possible mutagenic pathway for cells. 705 53

The replicon of a cryptic Thiobacillus intermedius plasmid (pTiK12) has been isolated and sequenced. Functional analysis of deletion subclones in Escherichia coli localized the replicon to a 3.5-kb region of DNA. Sequencing of this region identified a 30-bp A-T-rich potential stem-loop structure. In addition, an 11-bp direct repeat, an 11-bp inverted repeat, and a 16-bp inverted repeat were observed at the stem-loop structure. Also found in the replicon was a series of four tandem direct repeats consisting of a perfectly conserved 8-bp core. A region near the stem-loop structure is involved in the regulation of plasmid copy number. Deletion subclones lacking this region have increased copy numbers, indicating a negative regulatory role. An open reading frame capable of encoding a 320-amino-acid protein was found near the stem-loop structure. The putative amino acid sequence shares significant similarity with the two Rep proteins from the ColE2 and ColE3 replicons. Replication of the T. intermedius replicon is dependent upon DNA polymerase I. The isolation and examination of the T. intermedius plasmid replicon are initial steps toward the establishment of a genetic system in T. intermedius.
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PMID:Isolation and characterization of the replicon of a Thiobacillus intermedius plasmid. 775 4

CC-1065 is a minor-groove bonding agent capable of forming covalent adducts with the N-3 position of adenines within A-T-rich regions of duplex DNA. By examining the formation and location of CC-1065 adducts within the simian virus 40 (SV40) DNA molecule, the present study marks the first time that the precise sites of CC-1065 lesions have been identified at the level of eukaryotic genomic DNA. In naked DNA preparations, r values (moles of drug/mole of nucleotide base pair) > or = 0.0015 effected, after thermal treatment, a measurable decrease in intact supercoiled form I, as well as increases in forms II and III, indicating that both single-strand and apparent double-strand damage had occurred. A similar pattern of damage was observed in SV40-infected cells, albeit at higher CC-1065 levels. The amount of CC-1065 required to produce a 50% loss in form I was > 2-fold higher in infected cells (r = 0.029) than with purified DNA samples (r = 0.013). The appearance of double-strand damage at low drug levels suggested a high specificity of CC-1065 bonding to localized regions of the genome. The precise location of these CC-1065 adduction sites was examined by three methods: sequence analysis of the entire genome (GenBank), DNA polymerase termination assay of specific fragments of SV40, and restriction enzyme digestion analysis of the entire SV40 molecule. When sequence analysis of the entire genome was performed by examining both strands for the presence of the consensus CC-1065 binding sequence 5'-A/T-A/T-A/T-A/T-A*-3'[Reynolds et al. (1985) Biochemistry 24, 6228-6247], 294 single-strand adduction sites were predicted, compared to 20 sites where CC-1065 should bond to both strands within a 30-base-pair window and at which, when heated, a double-strand break should occur. DNA polymerase termination assay of actual adduction sites was performed on restriction fragments of SV40 DNA pretreated with CC-1065 in infected cells or in purified supercoiled DNA preparations and selected on the basis of the sequence analysis (i.e., regions 2510-2730, 3701-3920, 4400-4659, 4020-4320, and 5163-65). In general, double-strand lesions were detected in similar regions of the genome by the DNA termination assay and by sequence analysis. When restriction enzyme digestion and the DNA polymerase termination assay were compared throughout the genome, nearly identical patterns of adduct formation were observed. Interestingly, similar alkylation patterns were observed with either naked or infected cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:CC-1065 bonding to intracellular and purified SV40 DNA: site specificity and functional effects. 804 19

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from a poly [d(A-T)] template respectively 10- and 2-fold faster than the matched ones. Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same. The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.
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PMID:[Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha]. 838 31

Recent experiments have shown that difluorotoluene (F), a nonpolar isostere for thymine (T), codes efficiently and specifically for adenine (A) in DNA replication. F has almost the same shape as thymine but it is unable to form conventional hydrogen bonds with adenine. Therefore, it has been claimed that not hydrogen bonding but shape complementary may be important for the selection of the correct bases by DNA-replicating enzymes. In order to gain deeper insight into structure, charge distribution and energetics of the A-F and A-T base pairs we have performed quantum-chemical ab initio and density functional calculations at the HF, MP2 and B3LYP levels. The interaction energy of the A-F complex amounts to -3.8 kcal/mol (MP2) and is thus substantially smaller than typical ab initio interaction energies for Watson-Crick or non-canonical base pairs. The A-T and A-F complexes are planar and their overall geometries are similar (root-mean-square deviation: 0.4 A). The calculated donor acceptor atom distances in A-T are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. However, A-F shows a base pair opening as compared to A-T. Even though the interaction energy in the A-F base pair is small, the distances for the N6-H...F and N1...H-C3 contacts are still below the sum of the van-der-Waals radii, which means that the interaction is not governed by van-der-Waals forces alone. If the experimental findings can be confirmed, then our results indicate that DNA polymerase is able to retain high fidelity with base pairs of much smaller interaction energies than found for the conventional Watson-Crick and non-canonical base pairs.
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PMID:Quantum-chemical ab initio study on the adenine-difluorotoluene complex--a mimic for the adenine-thymine base pair. 944 8

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A >> T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.
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PMID:Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach. 1002 4

Although DNA polymerase fidelity has been mainly ascribed to Watson-Crick hydrogen bonds, two nonpolar isosteres for thymine (T) and adenine (A)--difluorotoluene (F) and benzimidazole (Z) --effectively mimic their natural counterparts in polymerization experiments with pol I (KF exo-) [JC Morales and ET Kool. Nature Struct Biol, 5, 950-954, 1998]. By ab initio quantum chemical gas phase methods (HF/6-31G* and MP2/6-31G**) and a solvent phase method (CPCM-HF/6-31G**), we find that the A-F interaction energy is 1/3 the A-T interaction energy in the gas phase and unstable in the solvent phase. The F-Z and T-Z interactions are very weak and T-Z is quite unstable in the solvent. Electrostatic solvation energy calculations on F, Z and toluene yield that Z is two times, and F and toluene are five times, less hydrophilic than the natural bases. Of the new "base-pairs" (F-Z, T-Z, and F-A), only F-A formed an A-T-like arrangement in unconstrained optimizations. F-Z and T-Z do not freely form planar arrangements, and constrained optimizations show that large amounts of energy are required to make these pairs fit the exact A-T geometry, suggesting that the polymerase does not require all bases to conform to the exact A-T geometry. We discuss a model for polymerase/nucleotide binding energies and investigate the forces and conformational range involved in the polymerase geometrical selection.
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PMID:Interaction and solvation energies of nonpolar DNA base analogues and their role in polymerase insertion fidelity. 1044 97


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