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)

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.
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PMID:An auxiliary protein for DNA polymerase-delta from fetal calf thymus. 374 89

An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.
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PMID:Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. 388 95

We identified by immunobinding assay the polypeptides synthesized as the result of amber mutations in the DNA polymerase gene of bacteriophage T5. Comparison of the size of such polypeptides revealed the order of mutagenic loci of these mutations and the direction of transcription of the gene. Extracts of cells infected with wild-type T5 and with five amber mutants of the polymerase gene (D7, D8, D9, am1, and am6) were prepared, and the proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After transfer of the proteins to a nitrocellulose sheet, a radioimmunolabeling technique was used to identify the T5 DNA polymerase and its amber mutant polypeptides. Based on the relative sizes of the polypeptides, the transcription of the T5 DNA polymerase gene was determined to proceed in the order D7, D8, am1, D9, and am6. The molecular weights of the DNA polymerase polypeptides coded by D8, am1, D9, am6, and T5+ were 23,000, 45,000, 75,000, 83,000, and 96,000, respectively. The D7-coded polypeptide was not detectable. These data suggest that the carboxyl-terminal region of the enzyme is essential for the polymerase function.
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PMID:Identification by immunobinding assay of the polypeptide coded by the DNA polymerase gene of bacteriophage T5 and its amber mutants and the direction of transcription of the gene. 389 73

A DNA polymerase with properties similar to mammalian polymerase beta has been isolated to near homogeneity from embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-gel electrophoresis indicates that this enzyme is composed of a single polypeptide of molecular weight-110,000. Optimum activity on a nicked template occurs at pH 8.4 in the presence of 15 mM MgCl2 and 250 mM NaCl. Enzyme activity is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmalemide. These properties clearly distinguish this enzyme from polymerase alpha, which has previously been characterized from this tissue. This report represents the first extensive purification of a beta-like polymerase from the Protostomic branch of the animal phylogenetic tree. It furthermore generates the potential for a genetic analysis of the function of polymerase beta in DNA recombination, repair, and synthesis.
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PMID:Purification and characterization of a DNA polymerase beta from Drosophila*. 392 21

DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.
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PMID:Inhibitory effects of various 3'-deoxyribonucleotides on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes. 408 76

The L-cell virion (LCV) has been purified from supernatant fluids of mouse L cells grown in suspension culture. The virion is similar to other RNA tumor viruses by several criteria: (i) the density of the virion is 1.16 g/cm(3); (ii) the virion appears as a rounded membranous particle with an outer diameter of 146.7 +/- 11.8 nm, and contains knobs (7-nm diameter) over its surface; (iii) 15 polypeptides (ranging in molecular weight from 7,000 to 110,000) are detectable after electrophoresis of virion protein in sodium dodecyl sulfate-polyacrylamide gels; (iv) three species of RNA can be isolated-high molecular weight (80 to 88s) (50%), 7s (35%) and 4s (15%); (v) heat denaturation of the high-molecular-weight RNA yields a heterogeneous population of molecules (20 to 35s) as well as a 7s and 4s species. Despite the general similarity to infectious RNA tumor viruses, LCV is apparently defective as evidenced by the fact that it does not induce tumors in animals or transform normal mouse cells in vitro (Kindig and Kirsten [17]). The defective nature of the LCV might be related to the fact that assays for DNA polymerase in the virion showed only a negligible activity when compared to Rous sarcoma virus.
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PMID:Physiocochemical studies on L-cell virions. 412 Jun 38

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.
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PMID:Structural studies of avian myeloblastosis virus: comparison of polypeptides in virion and core component by dodecyl sulfate-polyacrylamide gel electrophoresis. 412 94

Infectious hamster leukemia virus (HaLV) contains a DNA polymerase different from those of murine and avian viruses. No endogenous reaction directed by the 60 to 70S RNA of HaLV could be demonstrated in detergenttreated HaLV virions, nor could the purified DNA polymerase copy added viral RNA. The virion RNA could, however, act as template for added avian myeloblastosis virus DNA polymerase and the HaLV DNA polymerase could efficiently utilize homopolymers as templates. The HaLV enzyme was like other reverse transcriptases in that certain ribohomopolymers were much better templates than the homologous deoxyribohomopolymers. No ribonuclease H activity could be shown in the HaLV enzyme, but neither could activity be found in the murine leukemia virus DNA polymerase. The hamster enzyme was unique in that poly(A) .oligo(dT) was a poor template, and globin mRNA primed with oligo(dT) was totally inactive as a template. Its uniqueness was also indicated by its subunit composition; electrophoresis of the HaLV DNA polymerase in sodium dodecyl sulfate-containing polyacrylamide gels revealed equimolar amounts of two polypeptides of molecular weight 68,000 and 53,000. The sedimentation rate of the enzyme in glycerol gradients was consistent with a structure containing one each of the two polypeptides. The enzyme thus appears to be structurally distinct from other known virion DNA polymerases. Its inability to carry out an endogenous reaction in vitro might result from an inability to utilize certain primers.
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PMID:Hamster leukemia virus: lack of endogenous DNA synthesis and unique structure of its DNA polymerase. 413 18

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.
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PMID:Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose. 413 57

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.
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PMID:In situ activity of enzymes on polyacrylamide gels of a deoxyribonucleic acid-membrane fraction extracted from pneumococci. 415 52


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