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)

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.
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PMID:High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes. 116 71

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

DNA polymerase alpha from germinated wheat embryos was purified by ammonium sulphate fractionation, chromatography on DEAE-Toyopearl, followed by phosphocellulose and heparin Sepharose columns. The specific activity of the purified enzyme was more than 60,000 units/mg. It belongs to the alpha-type according to the large molecular mass, high sensitivity to NEM, aphidicoline, 200 mM KCl, low sensitivity to ethidium bromide and the absence of inhibition by ddTTP. DNA polymerase alpha consists of four subunits as shown by SDS-PAGE and seems to be homogeneous under non-denaturing conditions.
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PMID:Isolation of DNA polymerase alpha from germinated wheat embryos. 154 80

The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.
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PMID:Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase. 166 85

A small-scale plasmid preparation is described that is useful for a variety of procedures from double-stranded sequencing to in vitro transcription. No specialized equipment or reagents are required. The preparation of plasmid DNA does not require the use of RNase; instead the larger RNAs are precipitated with 2.5 M ammonium acetate. The resulting plasmid DNA is used routinely for double-stranded sequencing with the Klenow fragment of DNA polymerase and has been used for generating deletions with exonuclease III. In addition, the plasmid DNA has been used to generate transcripts with T7 RNA polymerase that translate well in reticulocyte lysate.
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PMID:A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription. 170 92

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

Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder.
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PMID:Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities. 193 78

The 21-tungsto-9-antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase alpha and murine DNA polymerase gamma. HPA23 inhibited the activity of DNA polymerase alpha in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template.primer. The Ki of murine DNA polymerase alpha for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase gamma also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to substrate, dTTP. DNA polymerase beta and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.
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PMID:Differential inhibition of various mammalian DNA polymerase activities by ammonium 21-tungsto-9-antimoniate (HPA23). 245 59

Phosphonoacetic acid (PAA) inhibits the replication of human herpesvirus-6 (HHV-6) in mononuclear cells from cord bloods which are susceptible for natural HHV-6 infection in humans. Nuclear extracts of uninfected or HHV-6-infected mononuclear cells were applied to phosphocellulose column chromatography, and DNA polymerase activity was measured with or without the addition of 100 mM ammonium sulfate. The major DNA polymerase activities eluted at 0.47 M KCl were suppressed in both uninfected and HHV-6 infected cells by the addition of 100 mM ammonium sulfate. DNA polymerase activity eluted at 0.47 M KCl was observed only from HHV-6-infected cells; it was enhanced by 100 mM ammonium sulfate and neutralized with immune serum. DNA polymerase activity eluted at 0.73 M KCl was determined to be HHV-6 specific and had the properties of a typical herpesvirus-induced DNA polymerase. PAA inhibited HHV-6-specific DNA polymerase activity.
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PMID:Phosphonoacetic acid inhibits replication of human herpesvirus-6. 256 36

A method for purifying T4 DNA polymerase from cells harboring overexpression plasmids is described. T4 DNA polymerase is precipitated from induced, lysed cells with polyethyleneimine, then extracted and fractionated further with (NH4)2SO4 before chromatography on a column of single-stranded DNA cellulose. This procedure can be completed in three days and consistently provides enzyme preparations which are at least 98% pure. When necessary, one further chromatography step provides T4 DNA polymerase suitable for recombinant DNA applications.
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PMID:Rapid purification of overexpressed T4 DNA polymerase. 262 73


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