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)

CMV has been reported to be associated with a DNA polymerase activity (DPA). In this communication its purification, characterization and potential diagnostic value were examined. CMV DNA polymerase was prepared from cell free supernatants of CMV (AD 169) infected cultures. Separation and purification of the enzyme was accomplished by column chromatography of the purified, lysed virus. CMV DPA was measured on an oligo (dT)-poly (dA) template primer. SDS-PAGE and western blot analysis under reducing conditions using an anti-CMV early antibody showed an 80 kDa protein band that was associated with the peak of polymerase activity. However, CMV isolates and CMV from urines from CMV retinitis patients immunoblotted by the same Ab revealed 140 kDa and 80 kDa bands under non-reducing and reducing conditions respectively, the latter was also associated with a 58 kDa band. The diagnostic value of the CMV associated DAP was tested using CMV positive urines. The latter demonstrated high PAA-sensitive DPA activity, compared to normal, HSV positive urines and urines from HBSAg positive patients. Taken collectively, these findings indicate the potential usefulness of CMV-associated DNA polymerase activity in the diagnosis and follow-up of patients with CMV-related illnesses.
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PMID:Cytomegalovirus DNA polymerase activity and an 80 kDa-associated polypeptide: a potential diagnostic tool for CMV disease. 818 15

We have identified a previously reported open reading frame (ORF13) that maps between pepA and valS at 96.6 centisomes of the Escherichia coli genome as the structural gene for the chi subunit of DNA polymerase III holoenzyme. This conclusion is supported by a perfect match of the amino-terminal 24 residues of chi with the DNA sequence of ORF13 and a demonstration that ORF13 directs expression of a protein that co-migrates with authentic chi on SDS-polyacrylamide gels. ORF13, designated holC, was isolated from the E. coli chromosome and inserted into a tac promoter-based expression plasmid to direct production of the chi subunit to 5-7% of the total soluble protein. The 3' end of holC was sequenced to resolve discrepancies between two published versions.
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PMID:Identification, molecular cloning and characterization of the gene encoding the chi subunit of DNA polymerase III holoenzyme of Escherichia coli. 824 93

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.
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PMID:Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta. 826 53

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase alpha (pol alpha) plays a relevant role in DNA synthesis and cell proliferation. Pol alpha gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer pol alpha antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 microM), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and cell count. [3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of pol alpha mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of pol alpha in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.
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PMID:Antiproliferative effect of DNA polymerase alpha antisense oligodeoxynucleotides on breast cancer cells. 850 May 51

A thermostable DNA polymerase from Thermus caldophilus GK24 was purified to near homogeneity by chromatographic methods, including ion-exchange, gel-filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75 degrees C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose-12 gel filtration. Reaction conditions were investigated in terms of pH, metal-ion concentration and temperature. Experimental results showed that T. caldophilus (Tca) DNA polymerase had a maximum activity near pH 8.7 at 75 degrees C. The N-terminal sequence of the enzyme was highly similar to that of Thermus aquaticus (Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5'-to-3' exonuclease activity and no 3'-to-5' exonuclease activity. Gene amplification using Tca DNA polymerase resulted in longer products than amplification using Taq DNA polymerase.
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PMID:Purification and characterization of Thermus caldophilus GK24 DNA polymerase. 850 85

Purification and characterization of a DNA repair enzyme having 5' apurinic/apyrimidinic (AP) endonuclease activity are reported. The enzyme extracted from 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) and single-stranded DNA cellulose, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme has an apparent molecular mass of 30 kDa as determined by SDS-PAGE. It was shown to have nicking activity on acid-depurinated DNA but not on intact DNA, and to have priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-treated DNA. The results indicate that it is a multifunctional DNA repair enzyme having 5' AP endonuclease and DNA 3' repair diesterase activities. The enzyme activity is dependent upon the presence of a divalent cation such as Mg2+. Its amino-terminal amino acid and internal amino acid sequences are determined.
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PMID:Purification and characterization of an AP endonuclease/DNA 3' repair diesterase from mouse ascites sarcoma cells. 854 4

A DNA polymerase was purified to near homogeneity from Trypanosoma cruzi epimastigotes. This preparation had a major polypeptide of 50 kDa and a minor band of 45 kDa. SDS-PAGE studies and a novel colorimetric activity gel technique demonstrated that the 50-kDa polypeptide chain is the catalytic subunit of this T. cruzi DNA polymerase. Western blot analysis of different purification stage fractions strongly suggests that this 50-kDa protein is the intact catalytic subunit and does not correspond to a degradation product from a larger one. This T. cruzi DNA polymerase is insensitive to aphidicolin, butylphenyldeoxyguanosine triphosphate, berenil, ethidium bromide and N-ethylmaleimide, but is markedly inhibited by the dideoxythymidine triphosphate analogue. Studies with different DNA templates showed that the DNA polymerase prefers activated DNA as substrate and that it cannot elongate oligoriboadenylate primers. The data presented in this paper are consistent with the hypothesis that this enzyme corresponds to a beta-like DNA polymerase present in the parasitic protozoon T. cruzi.
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PMID:Purification and characterization of a beta-like DNA polymerase from Trypanosoma cruzi. 857 47

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.
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PMID:In vitro reconstitution of human replication factor C from its five subunits. 869 48

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.
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PMID:Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. 874 Aug 35

Epstein-Barr virus (EBV) BALF2 gene product is one of the essential components in the lytic phase of the EBV DNA replication. The BALF2 protein was purified to near homogeneity from the nuclear extract of B95-8 cells with virus productive cycle induced by chemical agents. SDS-polyacrylamide gel electrophoresis showed the presence of a single polypeptide with a molecular weight of 130 K, which was identified as BALF2 protein by Western immunoblot analysis. On Superose 6 HR 10/30 gel filtration the BALF2 protein eluted at a position corresponding to an apparent molecular mass of approximately 128 K, indicating that the BALF2 protein behaves as a monomer in solution. The purified BALF2 protein bound to single-stranded DNA preferentially over double-stranded DNA or single-stranded RNA. Replication of singly primed M13 single-stranded DNA by the EBV DNA polymerase complex in the absence of the BALF2 protein exhibited a highly processive mode of replication and generated full length products in addition to some bands of pausing sites. Although the addition of the BALF2 protein did not affect the replication rate, the average chain length of the replication products was slightly increased with eliminating bands of pausing sites. Similar effects were observed with the reconstituted polymerase complex composed of the BALF5 and BMRF1 Pol subunits. On the other hand, in the absence of the BALF2 protein, the BALF5 Pol catalytic subunit alone extended the primer slightly and paused at specific sites on M13 ssDNA template where stable secondary structure is predicted. However, addition of the BALF2 protein, in contrast to the case of herpes simplex virus ICP8 which does not affect the overall distribution of length of the replication products synthesized by the HSV Pol catalytic subunit (Gottlieb et al., 1990, J. Virol. 64, 5976-5987), stimulated DNA synthesis and yielded a distribution of replication products with long lengths in addition to full length products. Although the BALF2 protein behaved as if it converts a low processive enzyme of the EBV Pol catalytic subunit to a highly processive form like the BMRF1 Pol accessory subunit, challenger DNA experiments revealed that the EBV Pol catalytic subunit is transferred to challenger DNA even in the presence of the BALF2 protein. It is therefore likely that the EBV BALF2 protein functions to melt out the regions of secondary structure on the single-stranded DNA template, thereby reducing and eliminating pausing of the EBV DNA polymerase at specific sites. These properties indicate that the EBV BALF2 protein acts as a single-stranded DNA-binding protein during lytic phase of EBV DNA replication.
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PMID:Epstein-Barr virus single-stranded DNA-binding protein: purification, characterization, and action on DNA synthesis by the viral DNA polymerase. 880 19


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