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)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins. A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase. The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis.
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PMID:Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus. 5 72

Unscheduled DNA synthesis was induced by bleomycin in isolated rat liver nuclei and in permeable mouse ascites sarcoma cells. ATP significantly enhanced the bleomycin effect of inducing unscheduled DNA synthesis. Replicative DNA synthesis in permeable mouse ascites sarcoma cells was inhibited by bleomycin. The apparent inhibition or stimulation by bleomycin of in vitro DNA synthesis was thought to be determined by a balance between inhibited DNA replicase activity and induced unscheduled DNA synthesis.
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PMID:Inhibition of replicative DNA synthesis and induction of unscheduled DNA synthesis in permeable sarcoma cell by bleomycin. 8 75

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.
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PMID:Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA. 12 75

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA-dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180 000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt-free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the gamma-position at a maximal rate near 10(4) molecules per enzyme monomer per min at 35 degrees C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single-stranded, circular DNA of phages phiX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein phiX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA-dependent ATPase of molecular weight 69 000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.
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PMID:Enzymic unwinding of DNA. 1. Purification and characterization of a DNA-dependent ATPase from Escherichia coli. 13 22

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.
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PMID:A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication. 13 39

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.
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PMID:Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition. 14 Jan 66

The first step in conversion of varphiX174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a varphiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.
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PMID:Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication. 14 14

An in vitro replication system reconstituted from six purified T4 bacteriophage proteins, each of which is essential for T4 DNA replication in vivo, requires ATP. Because of the complexity of the complete system, we examine in this report the involvement of ATP in two subsystems of the overall DNA synthesis reaction. One subsystem consists of the T4 DNA polymerase (gene 43 protein) and its "accessory proteins," the gene 44/62 and 45 products. An even simpler subsystem consists of the gene 44/62 and 45 proteins alone, which together have a DNA-dependent ATPase activity. The combination of the 44/62 and 45 proteins hydrolyze ATP to ADP and inorganic phosphate in the presence of DNA. These essential accessory proteins have been previously shown to increase T4 DNA polymerase activity on primed, single-stranded DNA templates. In this report we use nucleotide analogues to demonstrate that this polymerase stimulation requires hydrolysis of the beta,gamma-phosphate bond of ATP. However, our data suggest that the mechanism of accessory protein stimulation is such that less than 1 ATP molecule need be hydrolyzed per 10 deoxyribonucleotides incorporated by the DNA polymerase into DNA.
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PMID:An ATP stimulation of T4 DNA polymerase mediated via T4 gene 44/62 and 45 proteins. The requirement for ATP hydrolysis. 14 92

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells exhibited DNA excision repair when exposed to the following carcinogenic K-region epoxides: 7-methyl- and 7,12-dimethyl-benz[a]anthracene-5,6-oxide, chrysene-5,6-oxide and benzo[a]pyrene-4,5-oxide. This DNA excision repair was missing in uvr A and uvr B mutant cells. The K-region epoxide phenanthrene-9,10-oxide was ineffective in all E. coli strains tested. In contrast to the K-region epoxides which where found active only in wild type cells, 1,2,3,4-diepoxybutane and the 6,7-epoxides of the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate) elicited DNA repair in uvrA, uvrB mutant cells as well. Enzymic activities catalyzing particular repair steps were identified by determining a) repair polymerization and b) size reduction of denatured DNA. A) An easily quantifiable effect in E. coli wild type cells was epoxide-induced repair polymerization. None of the K-region epoxides tested stimulated DNA repair synthesis in uvrA, uvrB mutant cells, indicating that the uvrA-, uvrB-controlled UV-endonuclease initiated excision repair by cleaving epoxide-damaged DNA. 1,2,3,4-Diepoxybutane and the TPA-6,7-oxides induced DNA repair polymerization in uvr-deficient cells, although to a lesser extent than in wild type cells, suggesting the involvement of uvr-independent incision steps. None of the epoxides induced repair polymerization in a mutant (polA107) lacking the 5'--3'exonucleolytic activity of DNA polymerase I (exonuclease VI). The absence of any repair polymerization in the polA107 mutant indicates that the exonuclease VI plays a central role in removing epoxide-damaged nucleotides. As evidenced by greatly reduced levels of repair polymerization measured in polA1 cells, DNA polymerase I was the main polymerizing enzyme. b) As a consequence of treatment with 7-methyl-benz[a]anthracene-5,6-oxide, DNA from wild type cells, contrary to uvrA mutant cells, showed size reduction after denaturation and sedimentation in alkaline sucrose gradients. This is explained by repair-specific endonucleolytic cleavage of damaged DNA. The incision required the presence of ATP indicating that functional UV-endonuclease needs ATP as a cofactor.
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PMID:Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Analysis of DNA repair induced by carcinogenic K-region epoxides and 1,2,3,4-diepoxybutane. 15 97


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