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 cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)12--18 and poly(dC).oligo(dG)12--18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 micrograms/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)12--18 was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0.3 M with either activated calf thymus DNA or poly(dA).oligo(dT)12--18 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgCl2, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDS--polyacrylamide gel electrophoresis (SDS--PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.
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PMID:Human cytomegalovirus-associated DNA polymerase and protein kinase activities. 627 14

Seventeen out of 30 patients with chronic hepatitis type B with hepatitis B e antigen (HBeAg) in serum remained persistently positive for e antigen, while 13 seroconverted to antibody (anti-HBe) when followed over a period of one to five years. Initial levels of serum hepatitis B virus (HBV) markers, such as the hepatitis B surface antigen (HBsAg), HBeAg, and HBV-DNA polymerase (HBV-DNAP) were similar in the two groups of patients, while initial titres of the HBsAg-associated receptor for polymerized human serum albumin (pHSA), recently identified on HBV particles, were significantly higher in the patients who remained HBeAg positive (mean titre +/- SD = 2(-7.00) +/- 2(-3.2)) compared to the cases who eventually seroconverted to anti-HBe during the follow-up (2(-2.54) +/- 2(-2.14) P less than 0.001). A receptor titre above 1:64 by haemagglutination was highly predictive of persistence of HBeAg, suggesting that in patients with HBeAg-positive chronic hepatitis testing for the HBsAg-associated pHSA receptor may be useful in predicting the duration of HBe antigenaemia, with relevant clinical and prognostic implications.
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PMID:Virus receptors for polymerized human albumin: a prognostic marker in HBeAg-positive chronic hepatitis type B? 629 60

Hyperthermia has been shown to inhibit the activity of DNA polymerase-beta when either the intact Chinese hamster cells or partially purified enzyme samples from CHO cells are heated (42.2 or 45.5 degrees C). However, the loss of activity from heating isolated enzyme samples can be either greater or less than the loss from heating intact cells. For example, treating cells with the membrane-active agent procaine-HCl greatly sensitizes the cells to heat-induced loss of enzyme activity but has no effect on the heat sensitivity of isolated enzyme. Furthermore, the heat sensitivity of the isolated enzyme depends greatly on the purification steps and can be reduced by heating the enzyme in the presence of bovine serum albumin, activated DNA, or Langendorf salts. These observations, considered in relation to others in the literature, suggest that heat inactivation of polymerase-beta occurs from heat-induced changes in the intracellular environment, which in turn modify the direct thermal denaturation of the enzyme within the cell.
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PMID:Effect of hyperthermia on isolated DNA polymerase-beta. 687 33

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase alpha. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10(8) nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs. The specific DNA polymerase alpha activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10(8) cells, it decreases in mature cells to 57 units/10(8) cells, and in cells from senescent animals to 36 units/10(8) cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.
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PMID:Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice. 743 1

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.
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PMID:Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus. 746 44

The influence, on silver staining, of proteins and restriction enzymes in polymerase chain reaction (PCR) products was studied in 12-20% polyacrylamide gels. For small DNA fragments (74, 41 and 33 bp) best results were achieved using 20% polyacrylamide gels. In 12% gels, restriction enzymes and also proteins used for PCR (bovine serum albumin, Taq-DNA polymerase) interfere with silver staining of nucleic acids.
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PMID:The influence of proteins on silver staining of nucleic acids following polyacrylamide gel electrophoresis. 749 34

A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K(aff)) to polyclonal human IgG was found to be 3.3 (+/- 0.4) x 10(8) M-1 for divalent ZZ and 2.0 (+/- 0.1) x 10(7) M-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.
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PMID:Competitive elution of protein A fusion proteins allows specific recovery under mild conditions. 807 29

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.
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PMID:Heat-mediated activation of affinity-immobilized Taq DNA polymerase. 910 27

A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I from Thermus aquaticus. The DNA polymerase (deltaTaq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the deltaTaq DNA polymerase, affinity-purified ABP-deltaTaq could be heat-eluted from HSA columns by incubation at 85 degrees C. To produce free deltaTaq DNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure deltaTaq DNA polymerase with full enzymatic activity.
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PMID:Production of a thermostable DNA polymerase by site-specific cleavage of a heat-eluted affinity fusion protein. 911 94

Affinity systems based on specific molecular recognition are valuable tools for detection, purification and immobilization of recombinant proteins. Here, novel multipartite affinity fusion vectors were assembled and investigated to allow flexible binding and elution conditions. The rationale for the assembly of different combinations of affinity domains was to take advantage of the wide variety of molecular interactions of these domains for purification, solubilization, detection and immobilization. In total, seven different affinity tags representing five different types of tag-ligand interactions were studied: (i) monoclonal antibodies-peptides (T7-tag and FLAG peptide); (ii) streptavidin-peptide (Strep-tag); (iii) hexahistidyl-metal ions (His6-tag; (iv) bacterial receptors-serum proteins (staphyloccal protein A-Fc and streptococcal protein G-serum albumin); (v) streptavidin-biotin (in vivo biotinylated peptide). Selected tags were evaluated for the production and purification of Escherichia coli DNA polymerase I (Klenow fragment). On the basis of the results, a vector (pAff2c) was assembled using a novel combination of affinity domains: (i) an in vivo biotinylated peptide; (ii) a His6 sequence, and (iii) a highly soluble serum albumin binding region. Using these three affinities, a wide variety of conditions can be employed for both the binding and the elution steps.
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PMID:Multiple affinity domains for the detection, purification and immobilization of recombinant proteins. 917 44


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