Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.
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PMID:Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA. 135 40

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.
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PMID:Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions. 137 54

Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP). The modified DNAs were used as templates for in vitro DNA synthesis. DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types of adducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase. In addition, all three types of adducts completely blocked replication of ss phi X174 in an E. coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage. Polyacrylamide gel electrophoresis of DNA fragments synthesized by E. coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template. However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-trifluoroacetyl-N-acetoxy esters) were frequently bypassed during replication of ss phi X174 in E. coli, though with different efficiencies: 1 out 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation. Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.
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PMID:N-acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded phi X174 in Escherichia coli. 158 87

Our recent efforts have been directed at the development of selective inhibitors of different classes of viruses, including adeno, pox, and herpesviruses [herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV)], (+/-)RNA viruses (reo- and rotavirus), (-)RNA viruses (influenza, parainfluenza, measles, respiratory syncytial, vesicular stomatitis and rabies virus) and retroviruses [i.e. human immunodeficiency virus (HIV), the causative agent of AIDS]. In this search, the following molecular targets were envisaged: for DNA viruses in general, the viral DNA polymerase; for herpes simplex virus and varicella-zoster virus, the viral DNA polymerase via a specific phosphorylation by the viral 2'-deoxythymidine (dThd) kinase; for (+/-)RNA and (-)RNA viruses, S-adenosylhomocysteine (SAH) hydrolase, a key enzyme in transmethylation reactions required for the maturation of viral mRNA; for retroviruses, reverse transcriptase as initiator of virus replication and/or cell transformation; and for several enveloped viruses (i.e. retro-, herpes- and rhabdoviruses), virus adsorption to the outer cell membrane. Several new compounds have been developed that appear to act at these targets: i.e. (E)-5-(2-bromovinyl)-2'-deoxyuridine [bromovinyldeoxyuridine (BVDU)] and derivatives thereof [i.e. carbocyclic BVDU (C-BVDU)] as well as derivatives of acyclovir (i.e. 8-substituted acyclovir derivatives) as inhibitors of herpesviruses; (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and other phosphonylmethoxyalkylpurines and -pyrimidines as inhibitors of DNA viruses and retroviruses; acyclic and carbocyclic analogues of adenosine [such as (S)-9-(2,3-dihydroxypropyl)adenine [S)-DHPA), carbocyclic 3-deazaadenosine (C-c3Ado), (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (AHPA) alkyl esters, neplanocin A, 3-deazaneplanocin A and the 5'-nor derivatives of neplanocin A and 3-deazaneplanocin A] as inhibitors of (+/-)RNA and (-)RNA viruses; 2',3'-dideoxynucleoside analogues as inhibitors of retroviruses; and sulfated polysaccharides (i.e. heparin, dextran sulfate, pentosan polysulfate, mannan sulfate), sulfated polyvinylalcohol and co-polymers of sulfated polyvinylalcohol with acrylic acid as inhibitors of retro-, herpes- and rhabdoviruses.
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PMID:Selective virus inhibitors. 169 49

A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.
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PMID:Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes. 171 Aug 77

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.
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PMID:A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization. 171 53

Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents.
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PMID:Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase. 172 Oct 50

Modification of M13mp10 single-stranded DNA with 5-hydroxymethylchrysene (5HCR) sulfate, the ultimate carcinogenic metabolite of 5-methylchrysene, resulted in formation of N6[(chrysen-5-yl)methyl]adenine and N2[(chrysen-5-yl)methyl]-guanine at the ratio of 2.7:1. Measurement of DNA synthesis using this modified template and E.coli DNA polymerase I (Klenow fragment) demonstrated that increasing levels of adducts caused a progressive decline in replication. Analysis of reaction products on DNA-sequence gels revealed DNA elongation to be arrested exclusively at adenine adducts in -AAAGGA- and -AACA- sequences.
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PMID:DNA polymerase action blocked by adenine adducts induced by 5-hydroxymethylchrysene sulfate. 177 44

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained by DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes' molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.
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PMID:Purification and some properties of Candida albicans DNA polymerases. 179 90

Epstein-Barr virus (EBV) DNA polymerase mediates viral DNA replication during the lytic phase of the EB virus life cycle. In order to characterize its enzymatic activities EBV DNA polymerase was purified more than 1200-fold from chemically induced B95-8 cells. One polypeptide with molecular weight of 110,000 corresponded to the predicted EBV DNA polymerase, whereas the other polypeptides did not. A 3'-to-5' exonuclease activity was copurified with the EBV DNA polymerase through the course of the purification. Unlike HSV DNA DNA polymerase, 5'-to-3' exonuclease activity was not associated with the EBV DNA polymerase on the final step chromatography of single-stranded DNA agarose column. The associated 3'-to-5' exonuclease activity was stimulated by ammonium sulfate like the polymerase activity. It exhibited DNA-dependent nucleotide turnover activity and preferentially excised a terminal mismatched nucleotide on hybridized polynucleotides compared to the correctly paired substrate, indicating that the 3'-to-5' exonuclease may play a role in proofreading in the polymerization process.
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PMID:Characterization of 3'-to 5'-exonuclease activity associated with Epstein-Barr virus DNA polymerase. 185 Sep 10


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