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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system. This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur. After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates. RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa. Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain. Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E. coli-derived RT. Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.
...
PMID:Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system. 769 Jun 27

TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity. Here we report that it also has in vitro oriT-endonuclease activity. TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.
...
PMID:Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388. 785 4

Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
...
PMID:Studies of gapped DNA substrate binding by mammalian DNA polymerase beta. Dependence on 5'-phosphate group. 802 71

Upstream regions containing a novel common 8-base pair (bp) palindromic sequence, 5'-TATCGATA (Drosophila DNA replication-related element (DRE)), are required for the high expression of Drosophila genes for DNA polymerase alpha and the proliferating cell nuclear antigen (PCNA) (an auxiliary protein for DNA polymerase delta). Three DREs and one DRE are present in the DNA polymerase alpha gene (nucleotides-217, -83, and -30 with respect to the transcription initiation site) and in the PCNA gene (nucleotide-100), respectively. Deletions or 2-bp insertional mutations of DRE sequences led to an extensive reduction of promoter activities of both genes. Chemically synthesized oligonucleotides containing DRE sequences greatly stimulated the activity of the heterologous promoter of the Drosophila metallothionein gene, in addition to the promoter of the PCNA gene, when they were placed upstream from these promoters in a normal or a reverse orientation. The stimulatory effect increased synergistically and depended on the number of DREs. DRE activated the promoter when placed within 1.4 kilobases upstream from the promoter, but was much less active when placed 2.5 kilobases or more apart from the promoter. Using a gel mobility shift assay method, we obtained evidence for a protein factor (DREF) in the nuclear extract of cultured Drosophila cells (Kc cells), and this factor specifically binds to DREs of both genes. DNase I footprinting analysis indicated that DREF binds to the 24-bp DRE region of the DNA polymerase alpha gene in which 8-bp palindromic sequences are centered. A UV cross-linking experiment revealed that a polypeptide of approximately 90 kDa in the nuclear extract interacts directly with the DRE sequence. Using DRE-conjugated latex particles, DREF was affinity-purified from the Kc cell nuclear extract. By comparing results obtained by SDS-polyacrylamide gel electrophoresis and gel mobility shift experiments, we concluded that DREF is associated with the 86-kDa polypeptide. On gel filtration chromatography, a single peak of DREF activity was recovered in fractions corresponding to a molecular mass of 170 kDa, and the 86-kDa polypeptide was detected only in the corresponding fractions; thus, active DREF is probably a homodimeric form of the 86-kDa polypeptide. DREF may play important roles in coordinating expressions of Drosophila DNA replication-related genes.
...
PMID:Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase alpha and proliferating cell nuclear antigen. 809 16

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC1 purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28,000 and 30,000 Da, respectively. SDS-PAGE, in conjunction with activity gel analysis, gave a molecular mass of about 29,000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.
...
PMID:Isolation and characterization of endonuclease VIII from Escherichia coli. 811 Jul 59

In this study we report on the evidence that an alpha-like DNA polymerase purified from the thermoacidophilic archaeon Sulfolobus solfataricus has a modular organization of its associated catalytic activities (polymerase and 3'-5' exonuclease). This enzyme, a monomer of about 100 kDa whose complete primary structure is available, has a protease hypersensitive site that is likely to be cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE of highly purified S. solfataricus DNA polymerase samples. The amino-terminal microsequence analysis by Edman degradation has revealed that the 50- and the 40-kDa polypeptides correspond to the carboxyl- and the amino-terminal portion of the protein molecule, respectively. Using the bidimensional activity gel assay procedure, recently described by Longley and Mosbaugh (Longley, M. J., and Mosbaugh, D. W. (1991) Biochemistry 30, 2655-2664), we have demonstrated that the 50-kDa fragment retains a Mg(2+)-dependent DNA polymerizing activity, whereas the 40-kDa polypeptide is able to catalyze the excision of mispaired nucleotides at the 3'-OH terminus of a primer/template DNA substrate in the presence of Mn2+ ions. On the other hand, the 100-kDa protein possess both activities. To date, this is the first report indicating, on the basis of direct functional data, that the polymerization and the 3'-5' exonuclease activity of a family B DNA polymerase can be ascribed to physically distinct modules of the enzyme molecule.
...
PMID:Evidence that an archaeal alpha-like DNA polymerase has a modular organization of its associated catalytic activities. 813 6

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.
...
PMID:A meiotic DNA polymerase from a mushroom, Agaricus bisporus. 817 91

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.
...
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.
...
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.
...
PMID:Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta. 826 53


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---