Claude Houssier

Thermodynamics of Cation-Induced DNA Condensation

An estimation of the various free energy contributions to DNA collapse into toroidal particles is made, considering DNA bending and segment mobility, electrostatic repulsions between DNA chains, and attractive forces resulting from correlated counterion fluctuations. It is shown that the process of DNA condensation becomes spontaneous in the presence of divalent cations in methanol, and in the presence of tri- or tetravalent cations in water media. This is a consequence of the large decrease in the electrostatic repulsion between charged DNA segments, allowing the attractive force resulting from correlated fluctuations of bound counterions to become dominant. Our calculations indicate that short DNA fragments would condense into multimolecular particles in order to maximize the attractive force due to counterion fluctuations.

Tight binding of the antitumor drug ditercalinium to quadruplex DNA

The structural selectivity of the DNA‐binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H‐pyridocarbazole dimer was found to bind to double‐stranded DNA with a preference for GC‐rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four‐stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG3[T2AG3]3) provides new perspectives for the design of drugs that can bind to human telomeres.

Electric linear dichroism as a new tool to study sequence preference in drug binding to DNA

An original approach using electric linear dichroism (ELD) and natural DNAs and polynucleotides of differing base composition has been developed with the aim to investigate the sequence-dependent recognition of DNA by drugs. Both intercalators and minor groove binders have been studied as well as certain hybrid molecules. The results indicate that the orientation of drugs upon binding to nucleic acids can change markedly according to the target sequence. Among the intercalators tested, only actinomycin D and hycanthone show a clear preference for GC- and AT-rich sequences, respectively. For minor groove binders, the linear dichroism showing a strong dependence on base composition of the DNA and polynucleotides is most pronounced. Netropsin and distamycin bind to DNA with a marked AT specificity. Hoechst 33258, berenil and DAPI exhibit positive and negative dichroism signals at AT and GC sites respectively, suggesting that at least two types of drug-DNA interaction are involved depending on the AT/GC content of the DNA. Further investigations using polynucleotides with inosine substituted for guanosine, and competition experiments with intercalative drugs suggest that Hoechst 33258, berenil and DAPI interact with GC sequences via a non-classical intercalation process.

Different Binding Modes of Spermine to A-T and G-C Base Pairs Modulate the Bending and Stiffening of the DNA Double Helix

The influence of base composition (and sequence) on the process of interaction between synthetic polynucleotides and spermine, has been investigated using ultraviolet (including second derivative) spectroscopy, and electric dichroism. Different binding modes of spermine to poly(dG-dC) as compared to A-T containing polynucleotides, were evidenced. An interaction with the N7 and O6 of guanine is probably partially involved in the former case while simple electrostatic interaction with the phosphate groups would dominate in the latter. In the intermediate binding range (spermine over DNA phosphate molar ratios Sp/P of the order of 0.1 to 0.2), the complexes with poly(dA).poly(dT) and those with poly(dA-dT) displayed an important contribution of a permanent dipole moment to the orientation mechanism, as detected by the application of bipolar pulses in electric dichroism experiments. Just prior to precipitation (at Sp/P slightly larger than 0.3), these polynucleotides show electric dichroism and relaxation times characteristics corresponding to toroidal particles formation resulting from a bending of their chains. This implies asymmetric binding to phosphate sites on A-T containing polynucleotides. At low Sp/P ratios, spermine induced a stiffening of poly(dG-dC). No influence of spermine on the orientation mechanism of this polynucleotide was detected for Sp/P values ranging from zero to 0.35. The spermine-induced bending of A-T rich regions thus appears to be essential for DNA condensation into toroidal particles.

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.

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 Mannings 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.

Dielectric constant and ionic strength effects on DNA precipitation

We have investigated the effect of different zwitterionic compounds on DNA precipitation induced by spermine4+. Glycine, beta-alanine, 4-aminobutyric acid, and 6-aminocaproic acid have shown an increasing capacity to attenuate DNA precipitation. This protection effect has been correlated with the dielectric constant increase of their corresponding solutions. Calculations based on these experimental data and counter-ion condensation theory have confirmed the importance of this parameter for DNA-ion interactions and precipitation mechanisms. We have also observed a resolubilization of DNA in the presence of 6-aminocaproic acid at high spermine4+ concentration and in the presence of glycine at high spermidine3+ concentration. This could be explained by an increase of screening effect with polyamine concentration.

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.

Electro-optical properties of nucleic acids and nucleoproteins: IV. Influence of base composition and methylation on the properties of DNA and DNA-proflavine complexes

Abstract The electric dichroism, electric birefringence and relaxation times of calf-thymus and Micrococcus lysodeikticus DNAs (high molecular weight and sonicated samples) and of their complexes with proflavine have been studied. The influence of the methylation has also been investigated. No appreciable influence of the base composition and of the methylation of DNA on its electro-optical behaviour has been detected. The orientation of the proflavine ring was found to be parallel to the DNA base planes for the strongly bound dye molecules, irrespective of the base composition and methylation. The limiting amount of proflavine molecules which can be bound with this preferential orientation is very close to the value expected from the model corresponding to the exclusion of adjacent binding sites. Upon complexation, the mean birefringence relaxation times do not vary in a coherent way for the high molecular weight DNAs. They increase with the amount of binding in different proportions for the sonicated calf-thymus and Micrococcus DNAs. For both samples, however, the increase of the relaxation time was much lower than that expected from the viscosity observations and from the intercalation model.

Effect of Uridine Dethiolation in the Anticodon Triplet of tRNA(Glu) on its Association with tRNA(Phe)

The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.