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)

Reaction intermediates formed during the degradation of linear PM2, T5, and lambda DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn(2+) (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or lambda DNA, which do not contain nicks. In the presence of Mg(2+), when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length lambda DNA, EcoRI restriction fragments of lambda DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3' --> 5' direction. If this is correct, and since only 5'-monophosphate nucleosides are produced, then HSV DNase should "activate" DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg(2+) or Mn(2+), activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3' --> 5' direction as well as in the 5' --> 3' direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner.
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PMID:Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus. 626 48

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.
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PMID:Identification and characterization of a DNase induced by Epstein-Barr virus. 629 37

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.
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PMID:Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity. 645 73

Cycloheximide reversal experiments in chick embryo fibroblasts and mouse L-929 cells indicate that the poxvirus-induced enzymes DNA polymerase and 'alkaline' DNase are immediate early gene products of the virus. In contrast to the vaccinia-WR-coded enzyme under conditions of immediate early gene expression the cowpox-virus-induced DNA polymerase is made only in very small amounts. The studies are consistent with the notion that all poxvirus-specific early proteins may be immediate early viral gene products.
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PMID:Synthesis of early vaccinia-virus-specific enzymes under conditions of immediate early gene expression. 647 64

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.
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PMID:Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha. 657 66

Highly purified preparations of eukaryotic DNA polymerase alpha have been shown to contain primase activity (Kaguni, L.S., Rossignol, J-M., Conaway, R.C. Banks, G.R., and Lehman, I.R. (1983) J. Biol. Chem. 258, 9037-9039; Yagura, T., Kozu, T., and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127; Shioda, M., Nelson, E.M., Bayne, M.L., and Benbow, R.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7209-7213). We have investigated the de novo synthesis of DNA by a primase-DNA polymerase alpha preparation isolated from human HeLa cells using the synthetic homopolymers poly(dT) and poly(dC) as templates. In the presence of poly(dT), synthesis of poly(dA) required ATP in addition to dATP while synthesis of poly(dG) in the presence of poly(dC) required GTP in addition to dGTP. The primase activity required a much lower GTP concentration (Km = 0.1 mM) than ATP (Km = 0.8 mM) for the synthesis of DNA. Guanosine 5'-O-(3-thiotriphosphate), 5'-guanylyl-beta, gamma-imidodiphosphate, and 5'-guanylyl methylenediphosphonate substituted for GTP but the corresponding ATP analogues did not substitute for ATP. Furthermore, ATP and ATP analogues inhibited the GTP-dependent reaction while GTP and GTP analogues inhibited the ATP-dependent reaction. DNase treatment of products labeled with [alpha-32P] GTP revealed that an RNA oligomer was covalently linked to newly synthesized DNA. Alkaline hydrolysis of these products yielded GMP and pppGp, indicating that the primer was initiated with GTP. Alkaline hydrolysis of [alpha-32P]dGTP-labeled products yielded 2'- and 3'-GMP showing that DNA chains are covalently linked to the 3' ends of RNA chains. The primase activity could not be separated from DNA polymerase alpha through a 200-fold enrichment involving phosphocellulose, DNA-cellulose, hydroxylapatite, DEAE-cellulose and glycerol gradient purification steps. However, primase activity was found to be less stable than DNA polymerase alpha activity under a variety of conditions.
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PMID:Purification of a primase activity associated with DNA polymerase alpha from HeLa cells. 674 57

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.
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PMID:Interactions of calf thymus DNA polymerase alpha with primer/templates. 747 73

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.
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PMID:A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro. 812 85

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, O.C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25 degrees C and to temperatures as high as 100 degrees C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.
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PMID:Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes. 838 89

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.
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PMID:cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen. 839 45


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