Binding of ethidium bromide to ribosomal RNA. Absorption, fluorescence, circular and electric dichroism study.

The interaction between ethidium bromide and ribosomal RNA has been studied by means of absorption, fluorescence, circular and electric dichroism measurements in the near ultraviolet and visible regions at low ionic strength (1 . 10(-3) and 6 . 10(-3). The results have been interpreted on the basis of a model of interaction involving the intercalation of the phenanthridinium ring of the dye in the double-stranded regions of the RNA molecule, resulting in an increase of the dye-dye interactions as compared to DNA, and a stiffening of the intercalation regions.

An electro-optical study of the mechanisms of DNA condensation induced by spermine.

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.

Interaction of ethidium bromide with ribosomes. Absorption, fluorescence, circular dichroism and sedimentation studies.

It is shown in this work that the binding of ethidium bromide to yeast ribosomes occurs through intercalation in the double-stranded rRNA regions and produces changes in the ribosomes structure, yielding unfolded subparticles, and even partial separation of proteins from rRNA at very high binding ratios. The addition of Mg2+ prevents these structural changes, probably by partial inhibition of the dye binding.

Denaturation level of DNA-Pt complexes evidenced by Tb3+ fluorescence enhancement and electric dichroism.

The Tb3+ fluorescence is greatly enhanced, as a result of binding of various platinum coordination complexes to DNA, as compared to native DNA. The largest enhancement is observed for cis-Pt(NH3)2Cl2 but the fluorescence intensity does not however reach the level attained for thermally denatured DNA. Diethylenetriamine-Pt(II) produces very little increase of Tb3+ fluorescence. The electric dichroism in the DNA absorption band drastically decreases upon binding of the various Pt compounds investigated except diethylenetriamine-Pt. The results are discussed in terms of the various modes of binding of Pt derivatives to DNA, particularly in relation to the level of denaturation of the double helix.

Terbium(3+) as a probe of nucleic acids structure. Does it alter the DNA conformation in solution?

At low ionic strength, Tb3+ binding strongly alters the secondary structure of DNA. Circular dichroism and electro-optical techniques are more sensitive than fluorescence to study these alterations in double-stranded DNA, at low Tb3+/DNA phosphate (I/P) ratios. Both techniques yield the following conclusion: as I/P is increased, native and sonicated DNA undergo a transition from the B- to psi-form, the latter being a compact structure characteristic of aggregated DNA. Our study of alkylated DNA establishes that the accessibility of N-7 guanine to Tb3+ is clearly required for structural alterations in an aggregated state to occur. The chelation of the phosphate group and of the N-7 guanine by Tb3+ simultaneously alters the geometry of the sugar-phosphate backbone and the stacking interaction between the bases in double-stranded DNA.

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes.

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r less than 0.1-0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Electric birefringence of DNA and chromatin. Influence of divalent cations.

The effects of divalent cations on the DNA and chromatin conformation have been investigated by electric birefringence and birefringence relaxation measurements at low and constant ionic strength (0.001). An important decrease of the intrinsic optical anisotropy of DNA has been found in the presence of Mn2+ and Cu2+, but not with Mg2+. A complex variation of the mean relaxation time with the ratio I/P of ion to DNA-phosphate molar concentration has been evidenced in the presence of Mn2+ and Cu2+, while the mean relaxation time monotonously decreased in the presence of Mg2+. These observations are interpreted in terms of a specific organization of DNA in a compact, rigid structure, in the presence of Mn2+ and Cu2+, and a non-specific coiling in the presence of Mg2+. Drastic conformational changes encountered by chromatin in the presence of Mg2+ and Mn2+ cations have also been evidenced through electric birefringence measurements. They are interpreted by the formation of a superhelical compact arrangement of nucleosome strings which yielded a reversal of the birefringence sign with respect to the negative anisotropy observed in the presence of Na+ ions. The removal of the histone H1 prevented the appearance of this quaternary structure. More extended fragments of the chromatin chain obtained by ECTHAM chromatography of sonicated chromatin could not afford such compact arrangements.

Condensation and precipitation of chromatin by multivalent cations.

The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.

Interaction of DNA and purine nucleosides with cis-dichlorodiammineplatinum(II) and antimitotic activity of the complexes on meristematic root cells.

The appropriate experimental conditions for the preparation of complexes of cis-dichlorodiammineplatinum(II) with DNA and with purine nucleosides have been determined which leave negligible amounts of free drug in the solution. Important conformational changes of DNA upon binding to cis-Pt(NH3)2-Cl2 have been evidenced through viscosity, electric birefringence and thermal denaturation experiments. The antimitotic and antitumor activity of the drug was found to be totally inhibited by its binding to DNA and to the purine nucleosides. Enzymic degradation observations on the DNA-cis-Pt(NH3)2Cl2 complexes indicated an important inhibition of the degradation and the absence of release of free drug. The implications of the results in relation with the mode of binding of this compound to DNA, with the choice of carriers for drugs and the mechanism of action of lysosomotropic agents are discussed.

Preparation and some properties of strongly acidic proteins from calf-thymus nucleohistone.

The isolation of acidic proteins from calf-thymus nucleohistone (starting from purified nuclei) is reported. The method involved dissociation in 1 M KCl solution. Denaturating agents were not used at all. After electrophoresis on polyacrylamide gel, fractions containing a small number of components were obtained. The fractions display high ratios of acidic to basic amino acids, the ratios ranging from 4.0 to 1.5. In all fractions, the major components were of molecular weights in the ranges 12000-15000 and 24000-28000 as determined by gel-disc electrophoresis in dodecylsulphate and by equilibrium ultracentrifugation. Minor components of high molecular weights were also present. Amino-acid analyses are also reported. The tryptophan content was determined by a fluorometric method. Circular dichroism spectra depict a very low content of alpha-helicity that did not increase at higher ionic strength. A marked RNA-polymerase activity was found in one fraction.