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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized. This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I. Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP. DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted after reduction of the disulfide bond by dithiothreitol. The ability to recover biotinylated DNA from an avidin affinity column under nondenaturing conditions should prove useful in the isolation of specific protein-DNA complexes. As a demonstration of this approach, Bio-SS-DNA was reconstituted with histones to form 11S monomer nucleosomes. Bio-SS-nucleosomes were shown to selectively bind to avidin-agarose. Ninety percent of the bound Bio-SS-nucleosomes were recovered from the affinity column by elution with buffer containing 50-500 mM dithiothreitol. The recovered nucleosomes were shown to be intact 11S particles as judged by velocity sedimentation in a sucrose gradient. This approach may prove to be generally useful in the isolation of protein-DNA complexes in a form suitable for further analysis of their native unperturbed structure.
...
PMID:A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns. 388 7

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).
...
PMID:[DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation]. 408 23

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.
...
PMID:The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin. 620 65

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.
...
PMID:Properties of herpes simplex virus type 1 and type 2 DNA polymerase. 625 Jun 18

Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, being, as expected, 40 times higher than for T-DNA. Likewise, the differences in the yield of the s.s.act.DNA or s.s.BU-DNA replication between both enzymes were negligible. In contrast, damaged native DNA was 6 - 30 times more extensively replicated by DNA polymerase beta than alpha. We propose that this is due to the greater ability of DNA polymerase beta compared with alpha to replicate single-stranded gaps, the presence of which is more likely in damaged BU-DNA than in T-DNA and act.DNA.
...
PMID:Evidence implying DNA polymerase beta function in excision repair. 625 46

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.
...
PMID:DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40. 629 44

The polymerization reaction catalyzed by Escherichia coli DNA polymerase I (Pol I) has been studied by using the homopolymer template-primer system poly(dA).oligo(dT). Isotope-partitioning experiments indicate that the reaction follows an ordered mechanism in which Pol I first combines with template-primer to form an E.poly complex followed by addition of MgTTP and catalysis. The parameters governing the binding of Pol I to the template-primer are kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, and KD = 2 X 10(-7) M. Efforts to demonstrate the catalytic competence of the binary E.MgTTP complex were unsuccessful. Following initiation of the catalytic cycle, Pol I catalyzes the incorporation of an average of 40-50 TTP molecules into polymer before dissociating from the template-primer. The processive nature of the polymerization reaction as reflected by the isotope-trapping time dependence can be accounted for by a model in which processive synthesis is treated as a simple partitioning between continued polymerization (kcat = 3.8 s-1, 22 degrees C) and dissociation of the enzyme from the template-primer under steady-state conditions (koffss = 0.1 s-1). The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 degrees C) exhibited a pre-steady-state burst consistent with two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k = 4.6 s-1) and the other which follows this step (k = 4.0 s-1). Binding of MgTTP to the E.poly complex was shown to be a rapid equilibrium step by steady-state isotope-partitioning experiments. This suggested that the first rate-determining step may be a first-order isomerization which follows the binding of substrates and precedes bond formation.
...
PMID:Elementary steps in the DNA polymerase I reaction pathway. 635 5

A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase. This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation. The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha. The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells.
...
PMID:Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody. 639 27

A procedure has been devised for the purification of intact DNA polymerase alpha from early embryos of Drosophila melanogaster. The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000. These are related antigenically to the alpha (Mr 148,000), beta (Mr 58,000), and gamma (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G. R., Boezi, J. A. & Lehman, I. R. (1979) J. Biol. Chem. 254, 9886-9892]. The alpha subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo. As in the previous preparation, the alpha subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme. The ratio of primase to polymerase remains constant throughout the purification. Thus, the primase is very likely an integral component of the Drosophila DNA polymerase alpha. The purified DNA polymerase-primase contains no detectable endo- or exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCl optima identical to those reported previously. In contrast, the Km for dTTP is 3.7 microM as compared with 17.5 microM for the previous enzyme. Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged.
...
PMID:Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster. 640 45

2'-Deoxy-lin-benzoadenosine has been synthesized via reductive deoxygenation of 2-(beta-D-ribofuranosyl)-8-(methylthio)imidazo[4,5-g]quinazoline. The 5'-mono-, 5'-di-, and 5'-triphosphates have been prepared by chemical and/or enzymatic methods. The 5'-diphosphate was found to be a substrate for phosphorylation by pyruvate kinase and was compared with various natural and extended substrates in kinetic assays. When 2'-deoxy-lin-benzoadenosine 5'-triphosphate was tested in a nick-translation experiment with Escherichia coli DNA polymerase I, a very low level of 32P incorporation from [alpha-32P]TTP into poly[d(AT)] was observed. Nearest-neighbor analysis indicated that the analogue was not significantly incorporated into internal positions in the polymer. In DNA-sequencing reactions, the analogue caused chain termination at adensine residues, although termination was less uniform and less efficient than that with 2',3'-dideoxy-ATP. These experiments show that lin-benzoadenine can form a widened Watson-Crick base pair with thymine. They strongly suggest, though they do not prove, that the enzyme is able to attach the analogue to DNA.
...
PMID:Synthesis and biochemical evaluation of 2'-deoxy-lin-benzoadenosine phosphates. 648 84


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---