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PubMed1984

Different ability of novobiocin and coumermycin A1 to interact with nucleic acids.

The possibility of two structurally related antibiotics, Coumermycin A1 and Novobiocin, to interact with nucleic acids was investigated. Only Coumermycin A1 was able to form complexes with DNA showing an apparent affinity constant comparable to that of the interaction with ribosomal RNA. A binding specificity for A + T complementary and repeating sequences was also exhibited by Coumermycin A1. In view of the different behaviour of the two compounds some considerations are made on their mode of action; although they are acting on the same target enzymes in Escherichia coli, they may affect the functions of eukaryotic cells through a different mechanism not equally specific and probably distinct for each of the two antibiotics.

Aminocoumarins↗
PubMed1983

Stimulation of calf thymus DNA alpha-polymerase by ATP.

ATP stimulates the activity of the A and C forms of calf thymus DNA alpha-polymerase on several natural and synthetic primer-templates. Stimulations ranging from 1.5- to 8-fold were observed on gapped bacteriophage fd replicative form DNA, poly(dA) x oligo(dT)10, and poly(dT) x oligo(A)10, at ATP concentrations of 1-5 mM. CTP, dATP, and dCTP can substitute for ATP but are less effective. The nonhydrolyzable ATP analogs, adenyl-5'-yl imidodiphosphate and adenosine 5'-O-(thiotriphosphate), and other deoxy- and ribonucleoside triphosphates are essentially inactive for stimulation. Stimulation does not result from polymerase-associated DNA primase activity. The size of products synthesized processively by each enzyme on the two homopolymer templates was determined by gel filtration of primers extended under conditions where the enzyme did not react with a given 3'-OH terminus more than once. The size of products synthesized by the A and D forms on poly(dA) x oligo(dT)10 increased by a factor of 3-6 in the presence of ATP. This suggests a direct effect of ATP on the primer elongation reaction. Finally, presynthesis incubation of enzyme plus template at 37 degrees C in the presence or absence of ATP demonstrates that ATP stabilizes the enzyme against breakdown.

Adenosine Triphosphate↗
PubMedDate not supplied

[Local mobility of the DNA double helix. Comparison of conformational analysis with experiments].

The conformational studies of the torsional, tensile and bending flexibility of DNA within the B-family of forms are reviewed. The calculated values are in agreement with the experimental data. It means that the basic mechanism of the double helix elasticity descends from the small amplitude torsional vibrations of atoms. The torsional flexibility of B-DNA depends on the nucleotide sequence, namely poly(dA) . poly(dT) is stiffer than poly(dG) . poly(dC) due to the interactions between methyl groups of thymines and (C2(1) H2-groups of the neighbouring sugar rings. It is found that the B leads to A transition increases the torsional stiffness of the double helix as well. Possible correlation between conformational changes in the adjacent nucleotides is discussed. The crystalline structure of the dodecamer CGCGAATTCGCG testifies to the anisotropic flexibility of DNA and confirms the model for DNA folding in the nucleosome by virtue of "mini-kinks". The bending stiffness of DNA presumably depends on the base sequence: purine-pyrimidine dimers prefer bending into the minor groove, while pyrimidine-purine dinucleotides are more easily bent into the major one. Fluctuations in B-DNA are compared with those in alpha-helix.

DNA↗
PubMed1980

Role of the sigma subunit of Escherichia coli RNA polymerase in initiation. I. Characterization of core enzyme open complexes.

Open complexes of Escherichia coli RNA polymerase core enzyme with a poly[d(A-T)]-poly[d(A-T)]template have been characterized and compared with the previously characterized holoenzyme open complexes on the same template (Hansen, U. M., and McClure, W. R. (1979) J. Biol. Chem. 254, 5713-5717). The open complexes were monitored by the abortive initiation synthesis of the dinucleotide pApU, which is catalyzed by both enzymes. The major differences between the two complexes were: 1) the Michaelis constant for UTP was 60 times higher for core enzyme than for holoenzyme, 2) the intrinsic binding constant of core enzyme to the DNA was 3 orders of magnitude less than that of holoenzyme, and 3) cooperative binding of 2 core units was required for activity. Thus, the presence of the sigma subunit significantly altered the nature and extent of open complex formation. The rate of formation of the open complexes, however, was rapid for both enzymes. Rifampicin is shown to have a slight stimulatory effect on the extent of open complex formation of the core enzyme.

DNA-Directed RNA Polymerases↗
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↗
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↗
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↗