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)

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.
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PMID:Distinctive properties of mammalian DNA polymerases. 28 7

The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of 3H-thymidine-5'-triphosphate (3H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in 3H-TTP labelling index (3H-TTP LI) was apparent together with an inhibition of 3H-leucine incorporation (3H-LEU LI) into lymphoblasts. A moderate decrease in 3H-TDR labelling index (3H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of 3H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vncristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in 3H-TTP LI, 3H-TDR LI and 3H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G1.
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PMID:Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine. 28 67

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

Homogeneous preparations of phage T7 gene 4 protein catalyze the hydrolysis of dNTPs and rNTPs to NDPs and Pi in the presence of single-stranded DNA. Synthesis on single-stranded DNA by T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) does not affect the hydrolysis of NTPs by the gene 4 protein. Gene 4 protein does not catalyze the hydrolysis of NTPs in the presence of duplex DNA, nor can T7 DNA polymerase use duplex DNA as a template. However, the two proteins together can replicate duplex DNA and, under these conditions, synthesis is accompanied by hydrolysis of NTPs. During synthesis on duplex templates in the presence of T7 DNA polymerase, gene 4 protein, dNTPs, and rNTPs, 4.2 NTPs are hydrolyzed for each dNMP polymerized. 2'3'-Dideoxy-TTP, an inhibitor of DNA synthesis, inhibits hydrolysis by the gene 4 protein during synthesis on duplex DNA, and beta, gamma-methylene-dTTP, an inhibitor of hydrolysis by the gene 4 protein, stops DNA synthesis on duplex DNA. The multiple activities of gene 4 protein are shown to reside in a single protein molecule.
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PMID:Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. 32 56

We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.
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PMID:Incorporation of phosphorothioate groups into fd and phi X174 DNA. 32 75

The chemical synthesis of 5-alkyl-dUTP-s and their participation as substrates in poly[d(A-6)] primed polymerization reactions with dATP by E. coli DNA polymerase I enzyme has been described. In comparison with dTTP, at saturating substrate concentrations, the rate of hypochromic effect was found to be 17.3% higher for dUTP and was lower by 27.4% for 5-ethyl-dUTP, 29.5% for 5-n-propyl-dUTP, 31.4% for 5-n-butyl-dUTP and by 85.0% for 5-n-pentyl-dUTP. No hypochromic effect could be observed, however, with 5-iso-propyl-, 5-tert.butyl- and 5-n-hexyl-dUTP-s. Polydeoxynucleotides have also been isolated from the reaction mixture and some of their structural properties determined.
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PMID:Modified polynucleotides. I. Investigation of the enzymatic polymerization of 5-alkyl-dUTP-s. 33 94

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A--T)n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA)n . (dT)10 and (rA)n . (dT)10 . (20:1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA)n . (dT)10 and somewhat less for (dA)n . (dT)10. The apparent Km for the primer (rA)n . (dT)10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent Km for dTTP. The influence of polyamines on both the 5' leads to 3' and 3' leads to 5' nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A--T)n and T7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of the activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.
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PMID:Influence of polyamines on the activity of DNA polymerase I from Escherichia coli. 36 Oct 88

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.
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PMID:Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I. 36 22

A method is described for distinguishing deoxyuridine and deoxythymidine di- and triphosphate pools. The method utilizes a DNA polymerase assay for triphosphate determination and a coupled assay in which the disphosphate is converted to its corresponding triphosphate by nucleoside-diphosphate kinase and the triphosphate is measured by the DNA polymerase assay. By including deoxyruidine-triphosphate nucleotidohydrolase in the reaction mixture, dUTP is removed as a substrate for the polymerase. By determining differences in labelled acid-insoluble product formed in the reaction it is possible to determine dUTP, dUDP, dTDP and dTTP pools. Ribonucleotide reductase activity was determined by converting either CDP or ADP to its corresponding deoxyribonucleoside disphosphate and then using the diphosphate assay described for deoxyribonucleoside pools.
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PMID:An enzymatic method for distinguishing deoxyuridine and deoxythymidine nucleotide pools and its application for determining ribonucleotide reductase activity. 39 11

The cellular content and transcription program of the chloroplast ribosomal RNA genes of Euglena gracilis Z have been determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ([3H]ctrDNA). Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E. coli DNA polymerase I with [3H]TTP as a substrate. The [3H] DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA. The rRNA-[3H]ct rCNA hybrid was isolated by chromatography on hydroxylapatite. The [3H]ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of [3H]DNA-DNA and [3H]DNA-RNA hybrids. [3H]ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0,4,8,12,24,48, and 72 h after the onset of chloroplast development. From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made. Chloroplast rRNA increases from 2 to 26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases two- to threefold. Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than twofold, while the number of copies of rRNA per cell increases sixfold. The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental.
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PMID:Expression of the chloroplast ribosomal RNA genes of Euglena gracilis during chloroplast development. 40 48


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