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)

Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized this latter two-step approach for maximal hybridization signal brightness using DNA probes labeled to varying degrees with different fluorescent dyes. Reverse transcriptase and DNA polymerase 1 efficiently incorporated aa-dUTP into DNA, and adjusting the aa-dUTP:dTTP ratio controlled the degree of substitution. With cDNA probes hybridized to dot blots, probes having approximately eight dyes per 100 bases gave the best sensitivity, irrespective of the dye label. alpha-Satellite probes generated by nick translation and hybridized to human chromosome spreads also showed that probes having approximately eight dyes per 100 bases provided the brightest overall signals. These data demonstrate that this labeling method generates highly sensitive DNA probes that are difficult to obtain by conventional direct incorporation approaches. The technique is inherently consistent and versatile by virtue of the efficient incorporation of primary amines and the reliable chemical labeling reaction.
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PMID:Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling. 1474 Apr 93

The trifunctional dinuclear platinum compounds 1,2/c,c [[cis-PtCl(NH(3))(2)]mu-H(2)N(CH(2))(6)NH(2)[cis-PtCl(2)(NH(3))]](+) and 1,2/t,c [[trans-PtCl(NH(3))(2)]mu-H(2)N(CH(2))(6)NH(2)[cis-PtCl(2)(NH(3))]](+) contain a monofunctional platinum coordination sphere linked to a cis-[PtCl(2)(amine)(2)] moiety. The compounds have been examined for their DNA binding and ability to induce covalent ternary DNA-protein cross-links. Comparison was made with representative bifunctional dinuclear platinum compounds [[PtCl(NH(3))(2)](2)mu-H(2)N(CH(2))(n)NH(2)](2+). DNA modified by the trifunctional compounds is able to bind and cross-link BamHI, a sequence-specific DNA-binding protein that recognizes the palindromic sequence GGATCC and also very efficiently binds and cross-links SP1, a sequence-specific Zn finger protein that induces a bend in the DNA upon binding. Two representative nonsequence-specific DNA-binding proteins, the Klenow fragment from DNA polymerase I and Klenow exonuclease minus (which has been mutated to remove the 3'-5' proofreading domain), both bind modified DNA and effectively cross-link to the DNA. Data from circular dichroism, inhibition of ethidium bromide fluorescence, interstrand cross-linking and unwinding assays are all consistent with (Pt,Pt) interstrand cross-links as the dominant lesion of trifunctional compounds and the most likely structure to form the ternary DNA-protein cross-links. In vitro transcription of RNA is inhibited by the platinum compounds and indicate G residues as primary binding sites. Binding to calf thymus DNA as assessed by differential pulse polarography is rapid and essentially quantitative. An increase in melting temperature of CT DNA adducted by the platinum compounds is observed at low salt concentrations but at high salt, modification results in a decrease of t(m). In summary, the trifunctional agents may find use as protein-targeting drugs and as probes for conformational effects on DNA-protein interactions.
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PMID:Trifunctional dinuclear platinum complexes as DNA-protein cross-linking agents. 1519 20

We demonstrate that a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and accurate method for monitoring the stage and completeness of enzymatic DNA syntheses. A crucial step of these syntheses is to quench the reaction at the desired nucleotide length. This is especially important when expensive, e.g., (13)C/(15)N-labeled DNA segments, are synthesized for multinuclear magnetic resonance purposes to reveal detailed structural information. The analyses of three templates for a human telomeric 22-mer, a wild type, and a mutant human c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3'-5' exo(-) Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol. Small amounts of samples (ca. 1-2 microl each) were taken from the reaction mixtures at different times and analyzed promptly by MALDI-TOF, applying our successive on-plate desalting method that eliminates the insensitivity of the MALDI technique at high salt content. The progress of the reaction was detected by monitoring the relative intensity ratios of ions corresponding to the desired products and the primer-template complexes. The effectiveness of NH(3) cleavage leading to final products was also followed by MALDI-TOF in successful enzymatic reactions.
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PMID:Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protocol for monitoring the progress of enzymatic (13)C/(15)N-labeled DNA syntheses. 1598 27

Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the beta,gamma-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the beta,gamma-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3'N3), and ddTMP(3'NH2) from the 3'-end of primers with an equal efficacy.
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PMID:Hypophosphoric acid is a unique substrate of pyrophosphorolysis catalyzed by HIV-1 reverse transcriptase. 1627 6

The ends of linear chromosomes are capped and protected by protein-DNA complexes termed telomeres. Consequences of telomere dysfunction include genomic instability that can contribute to neoplastic transformation and progression. Telomere binding proteins interact with numerous proteins involved in DNA repair, underscoring the importance of regulating DNA repair pathways at telomeres. Telomeric DNA is particularly susceptible to oxidative damage, and such damage is repaired primarily via the base excision repair (BER) pathway. Using a screen for potential interactions between telomere repeat binding factor 2 (TRF2) and proteins involved in BER of oxidized bases in vitro, we found that TRF2 physically bound DNA polymerase beta (Pol beta) and flap endonuclease 1 (FEN-1). The interactions with endogenous proteins in human cell extracts were confirmed by coimmunoprecipitation experiments. The primary binding sites for both Pol beta and FEN-1 mapped to the TRF2 NH2-terminal and COOH-terminal domains. We further tested the ability of TRF2 to modulate BER protein partners individually on a variety of substrates in vitro. TRF2 stimulated Pol beta primer extension DNA synthesis on telomeric and nontelomeric primer/template substrates, resulting in up to a 75% increase in the proportion of longer products. TRF2 also stimulated Pol beta strand displacement DNA synthesis in reconstituted BER reactions and increased the percent of long-patch BER intermediates on both telomeric and nontelomeric substrates. Potential roles of TRF2 in cooperation with BER proteins for DNA repair pathways at telomeres, as well as other genomic regions, are discussed.
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PMID:Telomere repeat binding factor 2 interacts with base excision repair proteins and stimulates DNA synthesis by DNA polymerase beta. 1639 23

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.
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PMID:Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation. 1657 96

In order to learn more about the molecular basis for the inhibition of DNA replication produced by antitumor platinum drugs, we investigated DNA polymerization using DNA templates site-specifically modified with the 1,2-GG intrastrand cross-link of dinuclear bifunctional [{trans-PtCl(NH(3))(2)}(2){l-spermidine-N1,N8}](3+)(BBR3571) or conventional mononuclear cisplatin. These cross-links which have the same nature, but differ in the size and character of the conformational alteration induced in double-helical DNA, were analyzed for bypass ability with reverse transcriptase of human immunodeficiency virus type 1 and Klenow fragment of DNA polymerase I deficient in exonuclease activity. We found that the 1,2-GG intrastrand CL of BBR3571 inhibited DNA translesion synthesis markedly more than the same adduct of cisplatin. This result was explained by a larger size of the cross-link of BBR3571 and by a flexibility induced in DNA by this cross-link which can make the productive binding of this adduct at the polymerase site more difficult.
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PMID:1,2-GG intrastrand cross-link of antitumor dinuclear bifunctional platinum compound with spermidine linker inhibits DNA polymerization more effectively than the cross-link of conventional cisplatin. 1722 22

The use of cationic polymers for the delivery of DNA to mammalian cells has generated significant interest due to the intrinsic properties associated with these delivery vector systems. One particular system utilizing polyethylenimine (PEI) has been rigorously investigated. A major drawback associated with PEI is the cytotoxicity of the vector/delivery system. In an effort to combat this adverse side effect we have synthesized a novel random block copolymer based upon low molecular weight PEI. Here we report the grafting of Traut's reagent to residual primary amines of PEI to form a random block copolymer. The copolymer introduces a disulfide bridge upon oxidation of Traut's reagent capable of intracellular reduction. We confirm the successful grafting of this agent through FTIR and C-13 NMR. Molecular weight determination of the grafted copolymer was performed through HPLC-SEC and light scattering experiments. This polymer retains the ability to deliver GFP encoding plasmid DNA to 3T3 fibroblasts. Transfection levels were found to be approximately 90%. Transfection of T7 RNA dependent DNA polymerase was also performed utilizing our copolymer. We find successful activation of a virally introduced RNA transcript.
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PMID:Enhanced delivery of bioactive molecules to intracellular environments utilizing a cytosolically reducable grafted analog of polyethylenimine. 1745 Aug 55

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH(3))(2){d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin-DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients.
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PMID:Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase. 1849 11

We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr(4+) mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr(4+) links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH(3))(6)](3+), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.
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PMID:Electrochemical strategy for sensing protein phosphorylation. 2214 92


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