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 novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described. The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases. The modified nucleotide 9-(2-deoxy-5-O-triphospho-beta-D-ribofuranosyl)-N6,N6-ethano -2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2'-deoxyguanosine 5'-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase beta. Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage. The duplexes produced were found to be recognized as substrates by various DNA polymerases. The Km for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E. coli DNA polymerase I was found to be 29 microM, and the kcat was found to be 0.014 s-1. The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.
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PMID:A novel combined chemical-enzymatic synthesis of cross-linked DNA using a nucleoside triphosphate analogue. 198 67

A fraction (P1) which showed DNA polymerase III holoenzyme activity was obtained by partial purification including Polymin P fractionation from extracts of E. coli K-12 wild type (pol A+, pol B+) cells. The P1 fraction was composed of three macromolecular complexes, 11 S, 18 S and 24 S, all of which possessed holoenzyme activity. The activity of the P1 fraction was maximal at about 70 mM NaCl. The synthesis of long-chain poly(dT) with a poly(dA) oligo(dT)10 primer was dependent on the presence of ATP, but not on the presence of spermidine, suggesting that the single-stranded DNA binding protein (SSB) was present in the fraction. The intermediate lengths of the products in the absence of ATP and NaCl also suggest the functioning of DNA polymerase III'.
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PMID:DNA replication by novel macromolecular complexes involving DNA polymerase III holoenzyme activity. 634 Jun 88

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.
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PMID:High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. 832

DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase alpha was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [32P]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [32P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides synthesized using DNA polymerase from a 4 month-old rat. Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase alpha.
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PMID:Age-associated changes in the template-reading fidelity of DNA polymerase alpha from regenerating rat liver. 908 Mar 95

Taking advantage of strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications, a new electrochemical label-free integrated aptasensor based on silver microspheres (SMSs) as a separation element and graphene-mesoporous silica-gold nanoparticle (NP) hybrids (GSGHs) as an enhanced element of the sensing platform was first reported. In this sensing design (schematic representation of the sensing procedure for adenosine triphosphate detection, Scheme 1 in manuscript text), which contains an enhanced three-step magnification process, SMSs with "clean" surface were first used to separate the undesirable aptamer and aptamer-adenosine triphosphate (ATP) complex attached on SMSs surface after aptamer-ATP interaction, which lead to the detachment of blocker DNA into the solution phase. Then, under the assistance of blocker DNA, an amplified method based on the inherent signal-transduction mechanism of the hairpin probe and strand-displacement property of DNA polymerase was introduced. The obtained duplex DNA was used to hybridize with an acceptor DNA assembled on electrode to form triplex DNA, which could bring a more obvious detection signal compared with the duplex DNA without the amplification. The electrochemical signal came from the GSGH-based enhanced sensing interface containing positively charged ferrocene-appended poly(ethyleneimine) (Fc-PEI). Using the above multiple effects, we could achieve the sensitive analysis of a model small molecule-ATP (an important "molecular currency" of intracellular energy transfer) in a wide detection range from 0.05 nM to 56.5 nM with the detection limit of 0.023 nM.
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PMID:Solid-state label-free integrated aptasensor based on graphene-mesoporous silica-gold nanoparticle hybrids and silver microspheres. 2191 Apr 32