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)

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.
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PMID:Identification of DNA replication and cell cycle proteins that interact with PCNA. 939 13

The effects of bovine serum albumin, dithiothreitol, and glycerol on PCR were studied. PCR under standard conditions failed the amplification of an enterohemorrhagic E. coli DNA fragment when the boiled bacterial cell lysate was used as the template. The addition of either one of bovine serum albumin, dithiothreitol, or glycerol in the reaction mixture allowed the specific fragment amplification; and the optimum concentrations were as follows: bovine serum albumin, 1 mg/ml; dithiothreitol, 10 mM; and glycerol, 5%. In addition, when all of the three agents were included at the above concentrations, the PCR yield was further increased. The effect of the three-agent mixture was not the template specific. Furthermore, the mixture enabled long PCR over 20 kb when Taq DNA polymerase with 3'-5' exonuclease activity was used for the amplification. Our simple PCR method allows robust PCR independent of template purity or amplification length.
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PMID:Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR. 950 59

The short tandem repeats with repeat units ranging from two to several nucleotides became a powerful tool in the field of forensic identification and paternity determination as well as for research in human gene mapping. Allele and genotype frequencies for 9 short tandem repeats including HUMCSF1PO, HUMTH01, HUMPLA2A1, HUMF13A01, HUMCYAR04, HUMLIPOL, HUMHPRTB, HUMCD4, and HUMFABP were determined using PCR and subsequent analysis of the PCR products by denaturing polyacrylamide gel electrophoresis followed by silver-staining. DNA samples were obtained from about 100 Korean people and amplified in a thermocycler adopting glass capillaries rather than traditional tubes. We found that the bovine serum albumin was an essential additive for the capillary PCR, presumably to coat the inner surface of the capillary which may adsorb Taq DNA polymerase. The capillary thermocycler was very effective in reducing the cycling time such that most of the amplification reactions could be finished within 30 min albeit the PCR product was less than that for the tube systems. All loci except HUMHPRTB met the Hardy-Weinberg expectations according to the exact test. The cumulative power of discrimination (PD) was 0.9999998 and the power of exclusion (POE) for the paternity test was a little low, being 0.9873989.
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PMID:Survey of the Korean population for 9 short tandem repeat loci. 1059 42

Crude plant extracts were surveyed for their ability to inhibit DNA polymerase beta. A methyl ethyl ketone extract prepared from Baeckea gunniana was identified as a potent inhibitor of the enzyme. Bioassay-guided fractionation of the extract, using an assay to monitor the inhibitory potential of individual fractions toward DNA polymerase beta, led to the isolation of four active ursane and oleanane triterpenoids (1-4). Inhibitory principle 1 is a new natural product, and 2 is a novel compound. Their structures were established as 3 beta-hydroxyrus-12,19(29)-dien-28-oic acid (1) and 3 beta-hydroxyrus-18,20(30)-dien-28-oic acid (2) by spectroscopic analysis and by comparison with the data for the structurally related compound ursolic acid (4). Also isolated as a DNA polymerase beta inhibitor was oleanolic acid (3). Compounds 1-4 had IC50 values of 5.3-8.5 microM as inhibitors of polymerase beta in the presence of bovine serum albumin (BSA) and 2.5-4.8 microM in the absence of BSA.
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PMID:DNA polymerase beta inhibitors from Baeckea gunniana. 1065 12

Bioassay-guided fractionation of an active methyl ethyl ketone extract of Tetracera boiviniana, using a sensitive assay to monitor DNA polymerase beta inhibition, resulted in the isolation of three known triterpenoids, betulinic acid (1), 3-cis-p-coumaroyl maslinic acid (2), and 3-trans-p-coumaroyl maslinic acid (3). Compounds 1-3 inhibited DNA polymerase beta with IC50 values of 14, 15, and 4.2 microM in the presence of bovine serum albumin (BSA) and 6.5, 7.5, and 2.0 microM in the absence of BSA, respectively. Further, compounds 1-3 potentiated the effects of bleomycin in cultured P-388D1 cells.
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PMID:DNA polymerase beta inhibitors from Tetracera boiviniana. 1065 14

We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor that copurified with nucleic acids. Investigation of the candidate inhibitor melanin revealed that it potently blocks PCR at concentrations below 200 ng/ml, whereas 100 microg/ml melanin was required to inhibit reverse transcription. Melanin and thermostable DNA polymerase preferentially formed a distinct complex with reduced migration velocity as compared to pure polymerase in nondenaturating polyacrylamide gel electrophoresis. The inhibition of the enzyme by melanin could be reversed by diluting solutions of preformed complexes or by adding excess amounts of other proteins such as bovine serum albumin or dry milk. Our findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction.
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PMID:Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. 1081 30

In a survey of crude plant extracts for DNA polymerase 1 inhibitors, a methyl ethyl ketone extract prepared from Freziera sp. exhibited potent inhibition of DNA polymerase beta. Bioassay-guided fractionation of the extract, guided by an assay to detect DNA polymerase beta inhibition, resulted in the isolation of six active pentacyclic triterpenoids (1-6). These triterpenoids had IC50 values ranging from 7.5 to 16 microM in the presence of bovine serum albumin (BSA) and 2.6-5.8 microM in the absence of BSA, consistent with the possibility that these inhibitors may be of use in vivo.
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PMID:Pentacyclic triterpenoids from Freziera sp. that inhibit DNA polymerase beta. 1096 84

Bioassay-guided fractionation of extracts prepared from Brackenridgea nitida and Bleasdalea bleasdalei, using an assay to detect DNA polymerase beta inhibition, resulted in the isolation of the inhibitory principle, (24E)-3beta-hydroxy-7,24-euphadien-26-oic acid (1), a new euphane triterpenoid. The structure of 1 was established on the basis of HRMS and 1D and 2D NMR spectroscopic methods and was confirmed further by X-ray crystallographic analysis. Compound 1 inhibited rat DNA polymerase beta with an IC(50) value of 23 microM in the presence of bovine serum albumin (BSA) and 9.7 microM in the absence of BSA, consistent with the possibility that 1 may be of utility in vivo. This possibility was further supported by the finding that 1 potentiated the inhibitory action of the anticancer drug bleomycin in cultured P-388D(1) cells, reducing the number of viable cells by 48% when employed at a concentration of 25 microM in the presence of an otherwise nontoxic (75 nM) concentration of bleomycin. Compound 1 is the first euphane-type triterpenoid found to inhibit DNA polymerase beta.
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PMID:A new 7,8-euphadien-type triterpenoid from Brackenridgea nitida and Bleasdalea bleasdalei that inhibits DNA polymerase beta. 1107 51

The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.
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PMID:Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. 1110 81

The crystal structures and redox and UV-vis/EPR spectroscopic properties of two new mononuclear copper(II) complexes, [Cu(HL1)Cl2] (1) and [Cu(L1)Cl] (2), prepared through the reaction between copper(II) chloride and the ligand 2-[(bis(pyridylmethyl)amino)methyl]-4-methyl-6-formylphenol (HL1) under distinct base conditions, are reported along with solution studies. Also, we demonstrate that these CuII complexes are able to cleave unactivated peptide bonds from bovine serum albumin (BSA) and the thermostable enzyme Taq DNA polymerase at micromolar concentration, under mild pH and temperature conditions. The cleavage activity seems to be specific with defined proteolytic fragments appearing after protein treatment. The location of the specific cleavage sites was tentatively assigned to solvent-accessible portions of the protein. These are two of the most active Cu(II) complexes described to date, since their cleavage activity is detected in minutes and evidence is here presented for a hydrolytic mechanism mediating protein cleavage by these complexes.
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PMID:Hydrolytic protein cleavage mediated by unusual mononuclear copper(II) complexes: X-ray structures and solution studies. 1585 69


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