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)

The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected.
...
PMID:DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase. 78 35

A cellular factor which makes T7 DNA irradiated with gamma-rays a better primer for Micrococcus DNA polymerase was partially purified by DEAE and phosphocellulose column chromatography and named "primer activating enzyme". Sucrose density gradient sedimentation analysis was carried out to examine actions of one major active fraction that appeared by phosphocellulose chromatography. It was shown that this factor introduced new nicks in T7 DNA in addition to those introduced directly by gamma-ray irradiation. This enzyme fraction also had an endonucleolytic activity towards DNA containing apurinic sites induced by heat treatment and had capacity to enhance the priming activity of heat- or methyl methansulfonate-treated DNA but affected very little that of ultraviolet-irradiated DNA. This enzyme had no effect on T7 DNA when it was not treated with the DNA-damaging agents. From these results we concluded that this enzyme may be analogous to the endonuclease II or apurinic site-specific enconuclease of Escherichia coli.
...
PMID:Studies on DNA repair in Bacillus subtilis. II. Partial purification and mode of action of an enzyme enhancing the priming activity of gamma-irradiated DNA. 80 55

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.
...
PMID:Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli. 173 Jun 89

Sucrose density gradient analysis of Neurospora cell free extract showed at least three distinct peaks of enzyme activity; of these, a high molecular weight enzyme was identified as DNA polymerase alpha because of its sensitivity to aphidicolin and to NEM. DNA polymerase mutants of Neurospora crassa were isolated by their resistance to aphidicolin, a specific inhibitor of the eukaryotic DNA polymerase alpha. Some mutants showed an increase in the specific activity of the enzyme. One mutant (E-2-4-1) characterized in detail showed the presence of DNA polymerase which was resistant to inhibitory action of aphidicolin in an in vitro assay. Another mutant (C-3) showed changes in the pH optimum of the enzyme activity. Genetic characterization of the mutants provided evidence for the dominance of the aphr allele controlling aphidicolin resistance and its Mendelian segregation. Some of the aphidicolin resistant mutants were found to be UV-sensitive. Neurospora wild-type and mutant genomic DNA digest was found to hybridize with a cloned yeast DNA polymerase gene. The nick translated yeast DNA polymerase gene was used to screen a genomic cosmid library of Neurospora. A putative clone containing Neurospora DNA polymerase gene has been identified. Further molecular characterization of the Neurospora DNA polymerase gene and enzyme is in progress.
...
PMID:Characterization of eukaryotic DNA polymerases: aphidicolin resistant mutants of Neurospora with altered DNA polymerase. 214 49

The interactions of procainamide with DNA were studied by neutral and alkaline sucrose gradient sedimentation and sequential action of 2 enzymes: a mammalian repair endonuclease and bacterial DNA polymerase I. Sucrose gradient sedimentation shows that in the absence of photosensitization, the interaction of procainamide with DNA did not modify DNA sedimentation in alkaline or neutral sucrose gradients. In contrast, when a photosensitized DNA procainamide mixture was placed on sucrose gradients, the peak appearing on alkaline sucrose gradient after treatment with endonuclease was shifted toward the lower molecular weights, indicating that strand breaks had developed in the photosensitized procainamide DNA. Incubation of a [32P] labeled photosensitized procainamide-DNA complex with a repair endonuclease and DNA polymerase I released the label in the acid soluble fraction, indicating that only the photosensitized procainamide-DNA complex was susceptible to the endonucleolytic attack. There was only negligible release of the label in the acid soluble fraction without exposure of the DNA-procainamide mixture to light. The incorporation into DNA of [3H]-TTP (tritium labeled triphosphates) in presence of DNA polymerase I was inhibited when the photosensitized procainamide-DNA complex was used as substrate. However, after treatment of the photosensitized DNA complex with the repair endonuclease, the incorporation of [3H]-TTP was increased and reached values close to that observed with DNA unexposed to light, suggesting that the endonuclease functions as a repair enzyme.
...
PMID:Procainamide-DNA interaction. 283 1

Using hypophysectomized rats, it has been shown that DNA polymerase-beta activity in the adrenal gland and testis is largely influenced by pituitary trophic hormones. Sucrose gradient centrifugation of thyroid extracts revealed three peaks of DNA polymerase-beta activity sedimenting at 3.3S, 7.3S and 12S. Of these, hypophysectomy induced a decrease in the 3.3S DNA polymerase-beta, whereas other molecular forms were affected only slightly. DNA polymerase-alpha and -gamma activities were unaffected by hypophysectomy. These changes in DNA polymerase-beta caused by hypophysectomy were reversed by daily i.p. injection of TSH. Furthermore, stimulation of the thyroid by excess TSH induced by the administration of 1-methyl-2-mercaptoimidazole resulted in an increase of all forms of thyroid DNA polymerase-beta. These results show that the level of DNA polymerase is relatively constant after hypophysectomy but that DNA polymerase-beta in the rat thyroid gland is also modulated by TSH mainly through the change of activity of the polymerase-beta which sediments at 3.3S.
...
PMID:Hormonal regulation of DNA polymerase-beta activity in the rat thyroid gland. 319 62

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Binding of the DNA polymerase alpha-DNA primase complex to the nuclear matrix in HeLa cells. 367 71

The cell-free synthesis of DNA polymerase in translation mixtures containing calf thymus total and poly (A+) RNA was examined using activity gel analysis and immunobinding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that functional DNA polymerase catalytic polypeptides of Mr = 110,000 to 120,000 and Mr approximately 68,000 were synthesized. Sucrose density gradient centrifugation of total RNA resulted in resolution and partial purification of the mRNAs encoding these two DNA polymerase polypeptides. Immunobinding experiments with the monoclonal antibody to calf alpha-polymerase confirmed that an immunoreactive polypeptide of 110 to 120 kilo-daltons had been formed in vitro. This polypeptide and the 68,000-Mr polypeptide correspond in size to alpha-polymerase catalytic polypeptides observed in crude extracts of calf cells and in purified calf alpha-polymerase.
...
PMID:Synthesis of catalytically active polymerase alpha by in vitro translation of calf RNA. 608 7

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.
...
PMID:DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs. 632 Aug 90

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.
...
PMID:DNA polymerase I and DNA primase complex in yeast. 637 90


1 2 Next >>

---