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)

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.
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PMID:A DNA helicase from Xenopus laevis ovaries. 285 68

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).
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PMID:In vitro replication of DNA containing either the SV40 or the polyoma origin. 289 81

We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.
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PMID:Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. 289 82

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

Eleven simian virus 40-transformed cell lines from 5 different species were tested for their ability to amplify integrated simian virus 40 DNA upon infection with herpes simplex virus type I or treatment with various chemical carcinogens. Four cell lines were positive only for virus-induced gene amplification and two lines were positive for both carcinogen- and virus-induced gene amplification. Individual cell lines were assayed for the presence of an intact SV40 origin of replication, the expression of a functional SV40 T-antigen, and permissivity to herpes simplex virus replication. These parameters were found to be positive in all 6 amplification-competent cell lines. The ability of herpes simplex virus to amplify SV40 DNA sequences in transformed cells is greater than that of chemical carcinogens and can be suppressed by specific inhibitors of the herpes virus-encoded DNA polymerase.
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PMID:HSV- and chemical carcinogen-induced amplification of SV40 DNA sequences in transformed cells is cell-line-dependent. 298 11

Two forms of DNA-dependent ATPase activity were previously purified from the yeast Saccharomyces cerevisiae and characterized (Plevani, P., Badaracco, G., and Chang, L. M. S. (1980) J. Biol. Chem. 255, 4957-4963). Here, an additional DNA-dependent ATPase (ATPase III) has been purified from S. cerevisiae to near homogeneity. This ATPase differs from those described previously in its chromatographic properties, molecular weight, reaction properties and immunological relatedness. Its molecular weight is about 63,000 in the presence of sodium dodecyl sulfate. It hydrolyzes ATP to ADP and orthophosphate in the presence of DNA as an effector. In addition, yeast DNA polymerase I, which is a true DNA replicase of yeast, is stimulated severalfold by this ATPase. Neither yeast DNA polymerase II nor prokaryotic DNA polymerases are stimulated. This stimulation is intrinsic to the ATPase activity, since both activities copurified in the last four steps of purification, showed the same heat stability and showed dependence on and hydrolysis of ATP. The ATPase III preparation also contains a DNA-unwinding (DNA helicase) activity, which unwinds double-stranded DNA in the presence of ATP. In the S. cerevisiae radiation-sensitive mutant rad3, no significant ATPase III activity could be detected, suggesting that the RAD3 gene, which codes for a different polypeptide, regulates the expression of ATPase III activity.
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PMID:A new DNA-dependent ATPase which stimulates yeast DNA polymerase I and has DNA-unwinding activity. 301 45

We covalently bound periodate-oxidized ATP (oATP) to purified simian virus 40 (SV40) large T-antigen and determined the effect of this modification on viral DNA replication and three other biochemical activities of T-antigen. The oATP bound specifically to T-antigen, inhibiting the ATPase activity and preventing T-antigen from activating SV40 DNA replication in vitro. In contrast, binding of oATP had no effect on the DNA-binding activity of T-antigen nor on its ability to form a complex with DNA polymerase alpha. These results provide direct biochemical evidence suggesting that the T-antigen ATPase activity is necessary for viral DNA replication.
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PMID:Inhibition of simian virus 40 DNA replication by specific modification of T-antigen with oxidized ATP. 302 37

A purification procedure has been developed that resolves four chromatographically distinct DNA-dependent ATPase activities from calf thymus tissue. One of these activities has been purified to a nearly homogeneous protein, as judged by polyacrylamide gel electrophoresis. This protein has a specific activity of 18 mumol of ATP hydrolyzed per minute per milligram of protein and is active only in the presence of a DNA effector. The DNA-dependent ATPase activity is greatest in the presence of DNA containing a 3'-hydroxyl primer-template junction with a segment of adjacent single strand, i.e., a DNA polymerase substrate. Primer-template effectors that have had the 3'-hydroxyl group eliminated by the addition of a dideoxyribonucleotide are less active as cofactors for ATP hydrolysis than effectors which retain the 3'-hydroxyl group. Other DNAs can serve as cofactors, but with a reduced rate of ATP hydrolysis. DNA cofactors which are single stranded are much more effective at promoting ATPase activity than completely double-stranded cofactors, although the effectiveness of single-stranded DNA decreases as the length of the oligonucleotide decreases. An RNA/DNA hybrid does not promote ATPase activity. These data suggest that ATPase A may be involved in the recognition of primer-template junctions and the elongation phase of DNA synthesis.
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PMID:Purification of a calf thymus DNA-dependent adenosinetriphosphatase that prefers a primer-template junction effector. 302 61

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.
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PMID:Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. 303 60

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.
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PMID:DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization. 309 38


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