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)

A new method is presented for the determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate concentrations within human cells based on a DNA polymerase reaction directed by a palindromic oligonucleotide precursor. Two 19-mer oligonucleotide precursors are employed that contain a common 8-mer palindromic sequence followed by a sequence-specific insertion site and a 5'-oligodeoxythymidylate tail. To conduct a measurement, two molecules of the 19-mer oligonucleotide precursor are first annealed to form a pair of symmetrical template-primer addition sites at their 3'-termini that are coded for the analyte of interest, present in limiting amounts. The Klenow fragment of Escherichia coli DNA polymerase I then elongates the template-primer by the addition of two molecules of the complementary deoxyribonucleotide analyte. Following the addition of the analyte molecules, the template-primer is extended with a 10-mer oligo(dA) tail in the presence of excess dATP and the Klenow fragment. The result is a 30-mer palindromic oligonucleotide that can be separated from any remaining 19-mer precursor and quantified by paired-ion HPLC using UV detection. Since the molar extinction coefficient of the 30-mer palindromic oligonucleotide is much larger than that of the nucleotide analyte alone, the UV signal is markedly enhanced, thereby increasing sensitivity. Details describing this method and the application of it to measure these analytes in as few as 2.5 x 10(6) human cells are presented.
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PMID:Palindromic oligonucleotide-directed enzymatic determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate in human cells. 932 52

The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation.
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PMID:Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis. 939 59

We have developed a protocol for fast, nonradioactive, mRNA differential display reverse transcription PCR (DDRT-PCR) based on a commercial automated sequencer with RNA isolated from pig granulosa cells. We sought to discover conditions that would minimize the problem of using relatively small primers labeled with large infrared dye molecule, IR41, required for the sequencer. Extended IR41-labeled primers IR41-AAGC-T11-A, IE41-AAGC-T11-C and IR41-AAGC-T11-G gave more consistent differential display patterns than shorter anchored primers (IR41-T11A, IR41-T11C and IR41-T11G) without the additional (AAGC) cloning site. The optimal concentration of the extended labeled (downstream) primers was 20 pmol when 13-mer arbitrary (upstream) primers were used at a concentration of 4 pmol. Background smear and the intensity of amplified bands was significantly improved by changing from conventional Taq DNA polymerase to AmpliTaq Gold polymerase, which permits an improved "hot start" for the reaction. Running time (during which a digitized gel image is recorded) for a 26-cm polyacrylamide gel was 4 h, enabling us to analyze 90 reactions in an 8-h day. This protocol offers a rapid and reliable nonradioactive method for comparing gene expression patterns for various research or diagnostic purposes.
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PMID:Automated recording of RNA differential display patterns from pig granulosa cells. 945 67

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.
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PMID:Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I. 948 15

A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases. The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E. coli DNA polymerase I. The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases. The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone. Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer. PNA sequences were also recognized as template when annealed with a DNA primer. These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases. The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases.
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PMID:Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases. 948 18

8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases. The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair. Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation. Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation. Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively. Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue. Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair. Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.
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PMID:Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydroguanine base pair mismatches by bacteriophage T7 DNA polymerase exo-. 952 78

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.
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PMID:Evidence for reduced copying fidelity of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells. 962 Feb 26

Neither Sequenase 2.0 nor Klenow fragment were able to extend 12-mer primers using the eight templates (16-mers) derived by placing each of the four isomeric benzo[a]pyrene diol epoxide-deoxyguanosine adducts at the 13th nucleotide from the 3'-end of two different sequence contexts. Using an 11-mer primer to get a running start did not overcome the adduct induced block of primer extension except for the Klenow fragment and one of the two sequence contexts, indicating primer extension is dependent on both the polymerase and sequence context. In this case, purine nucleoside triphosphates (dATP>dGTP) were incorporated opposite each of the four adducts.
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PMID:Effect of single benzo[a]pyrene diol epoxide-deoxyguanosine adducts on the action of DNA polymerases in vitro. 966 21

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.
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PMID:Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs. 970 69

(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside phosphonate that has been shown to be effective in the treatment of AIDS although it has a shorter separation between the adenine and phosphorus than dideoxy-AMP and dAMP. By using pre-steady state kinetic methods, we examined the incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7 exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex, the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed by HIV-1 reverse transcriptase in that both analogs have similar substrate specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast, PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA 22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA (22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel studies will need to be performed using appropriate human polymerases, these results begin to define the mechanistic basis for the reported lower toxicity of PMPA in the treatment of AIDS.
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PMID:Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor, (R)-9-(2-Phosphonylmethoxypropyl)adenine. 976 48


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