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 protein that specifically enhances up to 13-fold the rate of copying of poly(dT) template by DNA polymerase alpha was partially purified from chromatin of regenerating mouse liver cells. This stimulatory protein, designated herein factor D, also increases 2-3-fold the activity of polymerase alpha with heat-denatured DNA and with primed, circular single-stranded phi X174 DNA. However, factor D has no detectable effect on the copying by polymerase alpha of poly(dG), poly(dA), and poly(dC) templates. Activity of mouse DNA polymerase beta is not affected by factor D with all the tested templates. In contrast to polymerase alpha, factor D is resistant to inactivation by N-ethylmaleimide and calcium ions, but it is readily heat-inactivated at 46 degrees C and is inactivated by trypsin digestion. Partially purified factor D is not associated with detectable activities of DNA polymerase, DNA primase, deoxyribonucleotidyl terminal transferase, and endo- or exodeoxyribonuclease.
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PMID:A DNA template recognition protein: partial purification from mouse liver and stimulation of DNA polymerase alpha. 409 24

Human placental extracts contain a specific inhibitor of mammalian retroviral RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) activity. This inhibitor copurifies with retrovirus-like particles in human placental extracts. The inhibitor can be removed from these particles by salt extraction, which leads to the recovery of the polymerase activity. Thus, the inhibitor does not irreversibly inactivate the particle-associated RNA-directed DNA polymerase activity. The inhibitory preparation contained no nuclease, protease, or phosphatase activity. Because its inhibitory action can be eliminated by the addition of more virus to the reaction, nonspecific inactivation of enzyme substrate has been ruled out. A partial characterization of the inhibitor indicates that it is (i) insensitive to ether, trypsin, and phospholipase C; (ii) stable to heat and pH 2-12; and (iii) nondialyzable.
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PMID:Human placentas contain a specific inhibitor of RNA-directed DNA polymerase. 616 15

A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.
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PMID:Stimulation of DNA polymerase alpha by a nuclear DNA/protein complex. 627 80

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.
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PMID:Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity. 645 73

A new purification technique for 'single-stranded DNA-binding proteins' from calf thymus permits the demonstration of a considerable heterogeneity within these proteins. Several molecular species are obtained with Mr between 24.10(3) and 30.10(3) and pI values between 6 and 8, showing significant differences with regard to the following functional properties: strength of binding to single-stranded DNA; lowering of melting temperature of poly[d(A-T)]; stimulation of DNA polymerase alpha on a poly[d(A-T)] template. Analysis of trypsin digestion products demonstrates that the different molecular species share extensive primary sequence homology. Experiments with antibodies show that the different molecular species are antigenically related and that a 31 kDa protein present in low amounts in our preparations is very cross-reactive.
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PMID:Structural and functional heterogeneity of single-stranded DNA-binding proteins from calf thymus. 653 27

A novel inhibitory factor which greatly inhibits DNA polymerase activity was isolated from the apical portion of the cauliflower inflorescence during purification of DNA polymerases. It can be adsorbed on a DEAE-cellulose column, but not on CM-Sephadex or DNA-cellulose. The factor exclusively inhibits the incorporation of [3H]dTTP into DNA when poly(rA, dT10) is used as the template primer, but not when activated DNA, heat-denatured DNA or native DNA is used as a template. The concentration of the factor in the reaction medium required for 50% inhibition is approx. 8 microgram/ml. The factor is heat-stable, is inactivated by trypsin, and has a maximum ultraviolet absorption at 278 nm. The molecular weight was estimated as 2500-3000 by Sephadex gel chromatography.
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PMID:Template-specific inhibitor for DNA polymerase isolated from cauliflower inflorescence. 721 35

A DNA polymerase was isolated from human spermatozoa. In one procedure, spermatozoa were decapitated with detergent, the heads purified and then lysed with dithiothreitol, trypsin and deoxyribonuclease. DNA polymerase was isolated from the lysate by sedimentation through an 18% Metrizamide solution, solubilization with 0.8 M-KCl-0.5% Triton X-100 and sequential chromatography on DEAE cellulose, phosphocellulose and hydroxylapatite. Alternatively, the heads of intact spermatozoa, untreated with detergent, were lysed as above; the subsequent Metrizamide pellet fraction was isolated and further fractionated by gel filtration and buoyant density centrifugation. The enzyme in this fraction was solubilized with KCl-Triton X-100. Characterization by velocity centrifugation and phosphocellulose chromatography revealed that it possessed properties indistinguishable from those of the enzyme purified from isolated sperm nuclei. The DNA polymerase had an apparent molecular weight of 79,000-89,000, Mn2+ (1 mM) was the preferred divalent cation and ativity was inhibited by concentrations of potassium phosphate greater than 10 mM. The synthetic template preferences of the enzyme were dT12-18 . poly rA > poly(dA-dT) > dT12-18 . poly dA; no activity was observed with dG12-18 . poly rC or dT10.
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PMID:Properties of a DNA polymerase from purified nuclei and DNA-synthesizing complexes of human spermatozoa. 743 Dec 98

The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3'-->5' exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3'-->5' exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an approximately 65-kDa proteolytic fragment of the alpha subunit retains the DNA binding function.
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PMID:Subunit structure of mitochondrial DNA polymerase from Drosophila embryos. Physical and immunological studies. 749 23

Herpesvirus DNA polymerases are prototypes for alpha-like DNA polymerases and important targets for antiherpesvirus drugs. We have investigated changes in the catalytic subunit of herpes simplex virus DNA polymerase following DNA binding by using the techniques of endogeneous fluorescence quenching and limited proteolysis. The fluorescence studies revealed a reduction in the rate of quenching by acrylamide in the presence of DNA without changes in the wavelength of the emission peak or in the lifetime of the fluorophore, consistent with the possibility of conformational changes. Strikingly, the proteolysis studies revealed that binding to a variety of natural and synthetic DNA and RNA molecules induced the appearance of a new cleavage site for trypsin near residue 1060 of the protein and increased cleavage by trypsin near the center of the protein. The extent of these cleavages correlated with the affinity of the polymerase for these ligands. These data provide strong evidence that binding to nucleic acid polymers induces substantial localized conformational changes in the polymerase. The locations of enhanced tryptic cleavage near sites implicated in substrate recognition and interaction with a processivity factor suggest that the conformational changes are important for catalysis and processivity of this prototype alpha-like DNA polymerase. Inhibition of these changes may provide a mechanism for antiherpesvirus drugs.
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PMID:Conformational changes induced in herpes simplex virus DNA polymerase upon DNA binding. 767 15

Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
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PMID:Studies of gapped DNA substrate binding by mammalian DNA polymerase beta. Dependence on 5'-phosphate group. 802 71


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