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)

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.
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PMID:Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases. 942 22

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase alpha-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase.
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PMID:Purification and properties of a DNA primase from Nicotiana tabacum. 944 86

The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase epsilon and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-linked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was 32P-labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The alpha, delta, epsilon and beta-like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by beta-poly(L-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase epsilon and a 135 kDa beta-like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei at growth arrest.
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PMID:Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling. 984 54

A series of 2-acetyl-pyridyl-4N-substituted thiosemicarbazones copper(II) complexes was evaluated for their cytotoxic mode of action in a variety of human and rodent tumor cell cultures. It was determined that these compounds may induce cytotoxicity by affecting several metabolic pathways including a reduction in de novo purine synthesis, and inhibition of IMP dehydrogenase, and DNA polymerase alpha activities. Selected compounds also demonstrated the ability to inhibit L1210 DNA topoisomerase II activity at micromolar concentrations. These agents were able to antagonize etoposide-induced formation of cleavable complexes as measured by K+/SDS precipitation and in vitro cleavage reactions.
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PMID:The cytotoxicity of copper(II) complexes of 2-acetyl-pyridyl-4N-substituted thiosemicarbazones. 989 58

In the early embryos of Drosophila, the B subunit of the DNA polymerase alpha-primase complex was found to migrate more slowly during the first 13 mitotic cycles than that from cycle 14 using SDS-polyacrylamide gel electrophoresis. Lambda phosphatase treatment showed that the reduced migration was caused by phosphorylation of the B subunit. Detailed analysis using the partially purified B subunit indicated that most of the B subunit until cycle 13 was a phosphorylated form while the B subunit of cycle 14 was a dephosphorylated form.
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PMID:Phosphorylation and dephosphorylation of the B subunit of DNA polymerase alpha-primase complex in the early embryogenesis of Drosophila. 991 45

The gene encoding Thermus filiformis (Tfi) DNA polymerase was expressed under the control of the tac promoter on a high-copy plasmid, pJR, in Escherichia coli. The Tfi DNA polymerase was purified by using heat treatment and DEAE-Sephacel column chromatography. The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE. The optimum pH and temperature of the enzyme were 8.4-9.0 and 70-72.5 degrees C respectively. The half-life of the enzyme at 94 degrees C was approx. 40 min. The enzyme was activated by the bivalent cations, Mg(2+) and Mn(2+), and was inhibited by EDTA. The optimal Mg(2+) concentration of the enzyme was 4 mM. The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg(2+) concentration. Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification. The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.
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PMID:Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli. 1046 14

Analogues of dUTP bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group linked via spacers of varying length (n = 2, 4, 7-13 atoms) to the 5-position of the uridine ring (NAB-n-dUTP) were synthesized and characterized. DNA polymerase beta efficiently incorporated these analogues into synthetic primer-template substrates in place of TTP, which allowed us to selectively introduce a photoreactive group at the 3' primer terminus. After completing photoreactive primer synthesis, the reaction mixtures were irradiated with monochromatic UV light (315 nm) in the presence of human replication protein A (RPA), a heterotrimer consisting of three subunits with molecular mass 70 kDa (p70), 32 kDa (p32), and 14 kDa (p14), and were separated by SDS-PAGE. The photoreactive primers cross-linked directly with p70 and p32, but cross-linking of p14 was not achieved even by varying the length of the spacer group. The data speak in favor of the protection of p14 by other RPA subunits from the interaction with 3'-end of the primer. Cross-linking of substrates to pol beta is inhibited when the analogue bears a short spacer (n = 2, 4, 7, and 8), but this is abrogated somewhat when longer spacers (n = 9-13) are examined. On the basis of these observations, we suggest that RPA and pol beta form a complex on primer-template substrates.
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PMID:Synthesis of base-substituted dUTP analogues carrying a photoreactive group and their application to study human replication protein A. 1089 64

DNA polymerase epsilon (Polepsilon) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS-PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Polepsilon. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Deltadpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Deltadpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Deltadpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Polepsilon in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S. cerevisiae Polepsilon is conserved in eukaryotes.
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PMID:Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae. 1102 62

The catalytic subunit of human DNA polymerase (pol) delta, p125, was expressed in recombinant baculovirus-infected insect cells, separated from a baculovirus-encoded DNA polymerase, and was purified to homogeneity by affinity trapping with a histidine-octapeptide at the C-terminus of p125 as the ligand. Purified p125 showed DNA polymerase activity resembling conventionally purified calf thymus pol delta. However, the two differed in four ways: 1) the specific activity of recombinant p125 was one quarter of the calf thymus pol delta; 2) the recombinant p125 was relatively resistant to aphidicolin; 3) the apparent Km for dTTP of the recombinant p125 was estimated at 33 microM, 15-fold the value for calf thymus pol delta; and 4) the recombinant p125 was not stimulated by recombinant PCNA, while activity of calf thymus pol delta increased 150-fold in response. Furthermore, PCNA did not stimulate either the p125 incubated with p50, a small subunit of pol delta, or co-expressed with p50 in insect cells. The full length recombinant p125 migrated slightly faster than pol delta from human cell lines, Jurkat or HeLa, upon SDS-polyacrylamide gel electrophoresis, suggesting a post-translational modification. The results indicate that in vivo assembly of the fully active complex of pol delta requires factors in addition to p125 and p50 subunits, and/or a post-translational modification of p125.
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PMID:Characterization of the human DNA polymerase delta catalytic subunit expressed by a recombinant baculovirus. 1120 90

In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.
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PMID:Phenotypic and genetic diversity of enterococci isolated from Italian cheeses. 1150 93


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