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At least 793 records · Page 44Linked to original sources
PubMed1980

Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. II. Release of sigma from ternary complexes.

The sigma subunit of Escherichia coli RNA polymerase was released from a transcribing polymerase-poly[d(A-T)]-poly[d(A-T)]-RNA complex when the nascent RNA reached a length of either eight or nine nucleotides. The dissociated sigma was separated from other reaction components by gel filtration, and was assayed using an activity assay previously described (Hansen, U. M., and McClure, W. R. (1979) J. Biol. Chem. 254, 5713-5717). The extent of RNA synthesis was limited by incorporation of 3'-dATP into the RNA chains; a series of distributions of product lengths was achieved by varying the concentration of 3'-dATP. A correlation of the number of moles of dissociated sigma versus the number of moles of RNA products of varying lengths allowed the determination of the point of release of the sigma subunit. Models to explain the cause of sigma release are discussed.

DNA-Directed RNA Polymerases↗
PubMed1981

On the fidelity of DNA replication. Nucleoside monophosphate generation during polymerization.

During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity. Using Escherichia coli DNA polymerase I and poly[d(A-T)] as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria. 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization. 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release. 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis. These observations argue against the kinetic proofreading mode to account for the fidelity of E. coli DNA polymerase I when copying poly[d(A-T)] in a Mg2+-activated reaction. Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step. However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro.

DNA Polymerase I↗
PubMed1982

Analysis of RNA polymerase by trypsin cleavage. Different structural changes produced by heparin and DNA.

Alterations in RNA polymerase structure can be detected using initial trypsin cleavage rates as a conformational probe. Both template (poly[d(A-T) . d(A-T)] and the RNA polymerase inhibitor, heparin, alter the rates at which the subunits of the enzyme are cleaved. However, while the presence of poly[d(A-T) . d(A-T)] slows the cleavage of subunits beta, sigma, and alpha by trypsin, heparin accelerates the cleavage of beta and sigma. Furthermore, the presence of heparin does not prevent the effect of poly[d(A-T) . d(A-T)] on the beta and sigma cleavage rates. Thus, heparin does not eliminate the interaction between DNA and RNA polymerase. That heparin does alter the nature of this interaction is demonstrated by the fact that template decreases the trypsin cleavage rate of subunit alpha in the absence, but not in the presence, of heparin. Like heparin, the addition of RNA to the reaction increases the accessibility of beta and sigma to trypsin. Hence the interaction of heparin with RNA polymerase may mimic the product, rather than the template, interaction.

DNA-Directed RNA Polymerases↗
PubMed1982

A study of the mechanism of T4 DNA polymerase with diastereomeric phosphorothioate analogues of deoxyadenosine triphosphate.

T4 DNA polymerase copolymerizes the SP isomers of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and 5'-O-(2-thiotriphosphate) with dTTP onto a poly(d(A-T) template in the presence of various metal ions. The corresponding RP diastereomers are inactive, independent of the metal ion used. The polymer resulting from the polymerization of the SP diastereomer of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and dTTP can be degraded by the 5' leads to 3' exonuclease activity of Escherichia coli DNA polymerase I and alkaline phosphatase (Brody, R. S., and Frey, P. A. (1981) Biochemistry 20, 1245-1252) to d(Tp(S)A). This material has the RP configuration as determined by comparison with the RP and SP diastereomers obtained by chemical synthesis and preparative separation by high performance liquid chromatography. This result indicates inversion of configuration at the alpha-phosphorus in the nucleotidyl transfer reaction and is compatible with the absence of a covalent enzyme intermediate.

Cations, Divalent↗
PubMed1982

Characterization of three subclasses of DNA-dependent RNA polymerases C from calf thymus.

DNA-dependent RNA polymerases from calf thymus tissue were solubilized and purified. Among seven chromatographically different forms of RNa polymerases obtained on DEAE-cellulose or DEAE-Sephadex chromatographies three subclasses of enzymes C were found. General enzymatic properties as: a moderate sensitivity to alpha-amanitin, divalent cation requirements, the preference for native DNA, efficient transcription of poly[d(A-T)], were similar to those described for C RNA polymerases from other sources. The molecular structures of CI, CII and CIII enzymes were indistinguishable; the enzymes were composed of two high molecular polypeptides 158,000 and 138,000 and several smaller subunits 99,000, 80,000, 78,500, 64,500, 52,000, 44,000, 32,000 and 24,000. Transcriptional activities of the chromatographically distinct forms of C polymerases were the same. The C enzymes exhibited striking affinity for homologous native A-T enriched DNA; Km values showed the greater number of binding and initiation sites localized in this template; therefore, A-T pairs are considered to be starter points for the transcription process catalysed by class C RNA polymerases.

Amanitins↗
PubMedDate not supplied

[Investigation of hydration of poly[d(A-T)] polynucleotide in helical and random coli states by the method of low temperature scanning microcalorimetry].

Thermodynamic parameters of the process of water phase transition in poly[d(A-T)] solutions in the wide range of polymer concentration have been studied. Curves of the heat capacity changes (delta Cp) depending on concentration of polymer and the temperature in the area of ice-water phase transition have been plotted. Thermodynamic parameters of the process of helix--random coli transition in water solutions of poly[d(A-T)] have been measured. Hydration (n) of poly[d(A-T)]: nAT = 28 mol H2O . mol-1 b. p. has been determined. Comparison of poly[d(A-T)] hydration with hydration of natural DNA shows that the hydration of AT-pairs exceed the amount of water, bound with GC-pairs (nGC congruent to 16--20 mol H2O . mol-1 b. p.). It was concluded that when describing the process of helix-random coil transition in DNA it is necessary to take into account not only the phenomenon of heterogeneity of stacking interactions, but also heterogeneity ("aperiodicity") in the structure of the hydrate shell of DNA molecules.

Base Composition↗
PubMed1977

On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase.

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.

Avian Myeloblastosis Virus↗
PubMed1987

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Alkylation↗
PubMed1981

Interactions of poly (N epsilon , N epsilon , N epsilon ,-trimethyllysine) and poly(lysine) with polynucleotides: circular dichroism and A-T sequence selectivity.

Complexes of (Lys) and [Lys(Me3)]n with natural and synthetic DNAs have been studied by CD as a function of ionic strength. In dilute EDTA, (Lys)n and [Lys(Me3)]n produce the same distortions to the CD spectrum of calf thymus DNA at r (peptide residue/nucleotide residue) values less than 0.6. At higher r values, the distortions are somewhat different. [Lys(me3)]n alters the conformation of some polynucleotides differently from (Lys)n under non- psi conditions. Therefore, methylation of histones may serve to alter the structure of chromatin. At low ionic strength, [Lys(Me3)]n and (Lys)n alter the viscosity of DNA to the same extent between r values of 0.0 and 1.0. In contrast to (Lys)n-DNA, at high ionic strengths, [lys(Me3)]n-DNA does not show psi - type CD spectra. (Lys)n forms psi - structures with (dA-dT)n and (dG-dC)n. [Lys(Me3)]n forms psi + structures with (dA-dT)n. Between 0.05 and 0.3 M NaCl, [Lys(Me3)]n forms psi+ structures with (dG-dC)n, while between 0.35 and 0.45 M NaCl, it forms a psi - structure with (dG-dC)n. Neither (Lys)n nor [Lys(Me3)]n forms psi structures with (dA)n.(dT)n or (dG)n.(dC)n. These results, in conjunction with the work of others on reconstitution of nucleosome-like particles from synthetic polynucleotides, suggest that the ability of DNA and histones to form nucleosomes is related to the formation of psi structures. (Lys)n binds preferentially to (dA)n.(dT)n over (dA-dT)n. [Lys(Me3)]n binds to (dA)n.(dT)n and (dA-dT)n with equal affinity.

Base Sequence↗
PubMed1987

Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site.

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and by calculating kD values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation. At 200 microM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase.

ADP Ribose Transferases↗
PubMed1990

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.

Adenosine Triphosphate↗