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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↗
PubMedDate not supplied

[Kinetics of formaldehyde splitting off from the hydroxymethylated amino groups of nitrogen bases incorporated into double-helical DNA].

We studied the kinetics of formaldehyde dissociation from hydroxymethylated amino groups of nitrous bases on native DNA. Compared with monomers, the rate constant of formaldehyde dissociation from such bases integrate in the double helix proved to be 20 times smaller for adenine and 4 times smaller for cytidine within the temperature range of 15 to 40 degrees C. The kinetic pattern suggests that the dissociation of formaldehyde from hydroxymethylated amino groups does not occur in the direction of the base plane nor through a full fluctuational opening of base pairs. It is presumed that formaldehyde dissociation from modified amino groups is due to softer fluctuational changes which however, make it possible for formaldehyde to attack amino groups perpendicularly to the base plane.

Chemical Phenomena↗
PubMed1980

Covalently bound ribonucleotides in crab d(A-T) polymer.

In addition to the known 3% of G + C residues, samples of purified crab d(A-T) polymer from Cancer borealis contained small amounts (< 3%) of RNA. Aliquots of d(A-T)n were digested with crude venom, and the resultant nucleosides were analyzed by high pressure liquid chromatography (HPLC); up to one-half of all guanosine was rG. Other aliquots were exhaustively digested with purified pancreatic DNase I to produce 88% dinucleotides. HPLC fractionation of this dinucleotide mixture into individual components revealed the presence of three mixed dinucleotides: -dC-rG, -dT-rA, and -dT-rG. A third aliquot of d(A-T)n was hydrolyzed overnight with 0.3 M KOH at 37 degrees C; approximately equal amounts of ribomononucleotides (predominantly containing purines) and deoxyribomononucleotides (predominantely containg thymine) were produced. KOH-hydrolyzable ribonucleotides accounted for one-third to one-half of the total RNA. The rest of the ribonucleotides remained with longer d-fragments, presumably as 3'(2')-terminal nucleotides (. . .d-d-d-rp). It was concluded that crab d(A-T) polymer from C. borealis contains 1 to 3% of dispersed, covalently bound ribonucleotides. The results also suggest that the sugar specificity of DNase I may be limited to a nucleotide following the cleavage.

Alkaline Phosphatase↗
PubMedDate not supplied

[Specific reaction between oligovaline and nucleic acids].

The DNA binding activity of trivaline dansyl hydrazide was investigated by circular dichroism, UV spectrophotometry and fluorescence methods. It is shown that these peptides in the absence of DNA can adopt statistical coil and antiparallel beta-conformation and can exist in aqueous solution as monomers, dimers and higher orders aggregates depending upon the concentration and the presence of N- and C-blocking groups. The aggregation and disaggregation processes are very slow especially for N- and C-protected peptides. Our observations show that oligopeptides in the monomeric and dimeric forms bind to double-stranded DNA and RNA whereas tetramers and higher order aggregates exhibit no DNA binding activity. The binding of monomers is a cooperative process favoring the formation of deformed antiparallel beta-structure between adjacently bound monomers. The binding constant of dimeric oligovaline species to GC-rich DNA sequences is about 5 fold higher than that found from the binding of oligovaline to poly(dA) . poly(dT). The binding of dimers takes place in the minor DNA groove as revealed from our observations that oligovaline binds to T6 phage DNA containing massive glucose and diglucose residues in the major groove. The backbone C=0 groups of oligovaline probably serve as specific reaction centres for the interaction with guanine 2-amino groups in the minor DNA groove.

Chemical Phenomena↗
PubMedDate not supplied

[Circular dichroism study of the reaction between the protein product of bacteriophage f1 gene 5 and synthetic polynucleotides].

The complexes of gene 5 protein (phage f1) with single-stranded and double-stranded polynucleotides were investigated by circular dichroism (CD). In our experiments the concentration of the protein varied accordingly to polynucleotide. A decrease of the CD amplitude for polynucleotide-protein complexes was shown in all the regions of wavelength studied. The data indicate that the protein is bounded to the polynucleotide. This protein bounds differently to double-stranded polynucleotides containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains. This difference is explained by differences in the form of the secondary structure of polynucleotides, containing ribo-ribo, ribo-deoxyribo and deoxyribo-deoxyribo chains.

Bacteriophages↗
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↗