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 soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.
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PMID:Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis. 111 87

Exposure of S-phase nuclei or subnuclear preparations from phytohemagglutinin-stimulated bovine lymphocytes to 0.02 M ATP caused an immediate and almost total loss of their ability to replicate DNA in vitro. Other ribonucleoside and deoxyribonucleoside triphosphates caused a similar inhibition of DNA replication. Levels of ATP which inhibit replication cause the release of DNA polymerases alpha and beta and small pieces of DNA from these nuclei. This release occurs both at 4 and 37 degrees C. The data support the conclusion that high levels of ATP or other nucleoside triphosphates inhibit DNA replication in nuclei by dissolution of the DNA replication complex. The limited success in reconstitution of the DNA replicase complexes is discussed.
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PMID:Dissociation of the DNA replicase system of bovine lymphocyte nuclei. 120 56

Epstein-Barr virus (EBV) DNA polymerase possesses a proofreading 3'-to-5' exonuclease activity (Tsurumi, T. (1991) Virology 182, 376-381). The 3'-to-5' exonuclease activity can be selectively inhibited by ribonucleoside 5'-monophosphates, while no inhibition of the DNA polymerase activity can be observed even when the template/primer concentrations are rate-limiting. Deoxynucleoside monophosphates except 5'dGMP have almost no effect on the exonuclease activity. Of the four ribonucleoside monophosphates, 5'GMP is the most potent (62% inhibition at 5 mM). The kinetic study shows that 5'-GMP inhibits the exonuclease activity competitively with respect to DNA template/primer. During DNA polymerization process the EBV DNA polymerase catalyzes the DNA-dependent conversion of complementary deoxynucleoside triphosphate to monophosphate form. With poly(dT).oligo(rA) as a template primer, selective inhibition of the exonuclease activity by 5'-GMP results in a decrease in the amount of free dAMP generated which is complementary to the template DNA, suggesting the functional relationship between the editing exonuclease activity and the chain elongation activity of the EBV DNA polymerase molecule.
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PMID:Selective inhibition of the 3'-to-5' exonuclease activity associated with Epstein-Barr virus DNA polymerase by ribonucleoside 5'-monophosphates. 132 5

The sugar boronated thymidine nucleoside, 5' -0-[(triphenylphosphine-boryl) carbonyl]-3'-0-acetyl thymidine 1, and the boron-modified nucleoside phosphotriester, 5'-(diethylphosphite- cyanoborane)-3'-acetylthymidine 2, were successfully synthesized. Both compounds demonstrated differential activity when tested against eight cell lines, with significant cytotoxic activity against the growth of human Tmolt3 leukemia, colon adenocarcinoma, HeLa S3 uterine carcinoma, and osteosarcoma cells. In in vivo studies these agents were found to be active against the growth of Ehrlich ascites carcinoma at 8 mg/kg/day I.P. and to be marginally active against the growth of L1210 and Lewis lung cancers in mice. The mode of action of these thymidine derivatives in Tmolt3 cells was the inhibition of DNA and protein synthesis. Compound 2 was highly effective in inhibiting DNA polymerase alpha and m-RNA, r-RNA and t-RNA polymerase activities. Both compounds inhibited ribonucleoside reductase activity. The de novo purine pathway appeared to be the major site of inhibition of the agents, with IMP dehydrogenase, PRPP amido transferase, and dihydrofolate reductase activities being significantly inhibited. In the pyrimidine pathway, carbamyl phosphate synthetase and aspartate transcarbamylase activities were inhibited by 1. As expected, d[NTP] levels were significantly reduced by treatment with the agents. DNA strand scission was evident after incubating Tmolt3 cells for 24 hr with the agents.
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PMID:Antineoplastic activity of boron-containing thymidine nucleosides in Tmolt3 leukemic cells. 150 1

Naturally occurring sesquiterpene lactones and their semisynthetic derivatives, such as the O = C-C = CH-bearing helenalin and its esters, have been shown to demonstrate potent cytotoxicity against the growth of murine L1210 lymphoid leukemia and human Tmolt3 leukemia, colon adenocarcinoma, HeLaS3, lung bronchogenic, KB, osteosarcoma, and glioma cells. The modes of action of helenalin in L1210 cells are the inhibition of DNA, RNA, and protein syntheses. This study confirms that thiol bearing enzymes of nucleic acid metabolism were significantly inhibited, e.g. DNA polymerase alpha, IMP hydrogenase, and ribonucleoside reductase. The addition of GSH to the reaction medium demonstrated total recovery of L1210 ribonucleoside reductase activity. Helenalin reduced cellular GSH levels in L1210 cells. Helenalin also reduced all four pool levels of d(NTP)s which would account for part of the observed inhibition of DNA synthesis. Reductions in the ribonucleotide pool levels were also generally evident after drug treatment. Thus, the sesquiterpene lactones appear to have more than one mode of action in L1210 cells. All of the modes of actions of helenalin are feasible mechanisms to lower nucleic acid synthesis and cause cell death of the L1210 leukemia cells.
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PMID:The cytotoxicity of helenalin, its mono and difunctional esters, and related sesquiterpene lactones in murine and human tumor cells. 152 2

The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.
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PMID:Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. 174 Apr 51

To investigate the role of the priming apparatus at the replication fork in determining Okazaki fragment size, the products of primer synthesis generated in vitro during rolling-circle DNA replication catalyzed by the DNA polymerase III holoenzyme, the single-stranded DNA binding protein, and the primosome on a tailed form II DNA template were isolated and characterized. The abundance of oligoribonucleotide primers and the incidence of covalent DNA chain extension of the primer population was measured under different reaction conditions known to affect the size of the products of lagging-strand DNA synthesis. These analyses demonstrated that the factors affecting Okazaki fragment length could be distinguished by either their effect on the frequency of primer synthesis or by their influence on the efficiency of initiation of DNA synthesis from primer termini. Primase and the ribonucleoside triphosphates were found to stimulate primer synthesis. The observed trend toward smaller fragment size as the concentration of these effectors was raised was apparently a direct consequence of the increased frequency of primer synthesis. The beta subunit of the DNA polymerase III holoenzyme and the deoxyribonucleoside triphosphates did not alter the priming frequency; instead, the concentration of these factors influenced the ability of the lagging-strand DNA polymerase to efficiently utilize primers to initiate DNA synthesis. Maximum utilization of the available primers correlated with the lowest mean value of Okazaki fragment length. These data were used to draw general conclusions concerning the temporal order of enzymatic steps that operate during a cycle of Okazaki fragment synthesis on the lagging-strand DNA template.
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PMID:Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. II. Frequency of primer synthesis and efficiency of primer utilization control Okazaki fragment size. 174 Apr 52

Silk gland cells of Bombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300,000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase alpha with no detectable level of polymerase beta. DNA polymerase alpha from silk gland extracts was purified to homogeneity (using a series of columns involving ion-exchange, gel-filtration and affinity chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a pI of 6.2 and the Km values for the dNTP vary over 5-16 microM. The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase alpha in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.
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PMID:Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori. 193 40

Purine and pyrimidine adducts of alpha-methylene-gamma-lactone demonstrated potent cytotoxicity against murine L1210 lymphoid leukemia growth as well as a variety of human tissue cultured tumors. The most potent compound, 9-[(2-methyl-4-methylene-5-oxotetrahydrofuran-2-yl)-methyl 1] adenine 1 demonstrated significant inhibition of DNA synthesis in L1210 leukemic cells with moderate inhibition of protein synthesis. The major enzyme activities inhibited by 1 were DNA polymerase alpha, ribonucleoside reductase and t-RNA polymerase with marginal inhibition of thymidine kinase, TMP kinase, PRPP amidotransferase and IMP dehydrogenase. The inhibition of DNA polymerase alpha activity by 1 was evident at the lowest concentration 25 microM and was evident within 15 min incubation at 100 microM. The magnitude of enzyme inhibition was consistent with the observed DNA synthesis inhibition by 1. The only deoxyribonucleotide level reduced by 1 was the dATP pool level. U.V. absorption of DNA after interacting with 1 demonstrated a hyperchromic effect and L1210 DNA strand scission was observed after 24 hr incubation with 1 suggesting some type of interference with the DNA template by the drug.
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PMID:The effects of alpha-methylene-gamma-lactone purines and pyrimidines on L1210 lymphoid leukemia nucleic acid metabolism. 201 69


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