Francesco Pedone

Temperature dependence of DNA dielectric dispersion at radiofrequency.

We have studied the dielectric behavior of DNA aqueous solutions at various ionic strengths and in the presence of the specific DNA ligand ethidium bromide, in the frequency range 1 MHz-1 GHz, at different temperatures ranging from 5 to 40 degrees C. The activation enthalpies of the dielectric relaxations studied were obtained by Arrhenius plots of In(tau T)-1 vs. T-1. The results are consistent with a counterion fluctuation model as previously developed by Mandel and colleagues.

Electrical conductivity and dielectric dispersion of E. coli 70S ribosomes and of 30S and 50S subunits: effects of magnesium ions

Abstract Electrical conductivity and dielectric dispersions have been measured on suspensions of 30S, 50S subunits and 70S ribosomal particles from E. coli, in the presence and absence of magnesium ions. Conductivity measurements show a net contribution due to the disperzed phase. Ribosomal particles act in general as obstructants to the ionic motions in the solution, but besides this effect, there is an intrinsic contribution to the overall conductivity which is more pronounced in the absence than in the presence of magnesium ions. This effect suggests the existence of an interfacial conductivity, modulated by bound magnesium ions. The intrinsic conductivity is higher in the 30S subunit than in the 50S. Dielectric dispersions show a consistent difference between 30S and 50S, indicating that the exposure of ribosomal RNA should be greater in the 30S than in the 50S. Structural information is derived from the conductivity measurement, via the Looyenga equation.

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences.

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.

Influence of defects on the electrophoretic, thermodynamic and dielectric properties of a 21 base pair DNA in solution.

The thermodynamic and dielectric properties of a 21 base pair DNA have been evaluated and compared with those of samples with some defects. In particular, fragments in which the absence of a phosphate group (nick) or of two nucleotides (gap) causes chain interruptions were studied. Measurements of ultraviolet absorption as a function of temperature at different oligomer concentrations and at various ionic strengths were performed. Dielectric spectroscopy at radiofrequencies (1 MHz-1 GHz) was applied on aqueous solutions of the duplexes at 5 degrees C, where the solutes are thermally stable. Dielectric dispersions with 30-40 MHz characteristic frequencies were defined. The results of melting experiments indicate a thermal destabilization of the oligomers containing the defects. Electrophoretic data and the dielectric results show that the conformations of the nicked and control samples are very similar, while the oligomer with a gap is more compact with a different charge distribution at the ends.

Effects of magnesium ions on ribosomes: A fluorescence study

Fluorescence intensity measurements of ethidium bromide (EB) bound to ribosomal RNA (rRNA) in suspensions of 30S and 50S subunits, of 70S ribosomal particles and of protein-free extracted rRNA are presented. Changes in the intercalation of EB reflect changes in conformation and degree of exposure of rRNA. The effect of removal of magnesium ions on the binding of EB is compared in protein-free rRNA and in ribosomal particles by a Scatchard plot analysis. In free ribosomal RNA the number of bound EBs do not depend on magnesium content, only the association constant is affected. In intact 70S particles and both in the separated 50S and 30S subunits the presence of magnesium greatly reduces binding of EB and no saturation of the fluorescence intensity with rRNA concentration is observed, preventing a Scatchard plot analysis. Removal of magnesium restores a strong EB intercalation. Then magnesium ions induce a conformational change in the 70S particles as well as in the separated subunits. The different behavior of the free-rRNA and of the ribosomal particles indicates that ribosomal proteins are relevant to the structural changes induced by magnesium ions. The comparison of the number of excluded sites and of the association constant in the 30S, 50S subunits and in the 70S particles indicates that even without Mg2+ ions the two subunits still interact, at variance with the commonly shared opinion that subunits dissociation takes place at low magnesium concentration.

Torsional constant of 27-mer DNA oligomers of different sequences

We have studied the torsional elastic constant (alpha) of short DNA (27mer) oligomers of various sequence by fluorescence polarization anysotropy (FPA) measurements. The lowest alpha values were found in samples with sequence rich in AA dinucleotides or containing the alternating d(A-T) x d(A-T) motif. The torsional rigidity of our DNA samples was compared to that calculated according to the current values of twist angle fluctuations derived for ten dinucleotide steps by recent analyses of DNA crystal structure database. The values of torsional rigidity derived from crystals are higher than our experimental ones, obtained by FPA analysis, suggesting that packing force in crystals may notably hinder the dinucleotide twist angle fluctuations that occur in solution. This behaviour is more evident for samples containing AA, TA and AT steps. In all the samples there is about a twofold change of the alpha value in the 10-40 degrees C range. An activation enthalpy (Delta H (#)) of about 17.4 kJ mol(-1), on average, was obtained for the temperature dependence of eight of the ten samples studied. A correlation with the stacking energy is discussed.

Radiofrequency dielectric spectroscopy of ribosome suspensions

Dielectric measurements on different ribosome suspensions were carried out in the frequency range from 10 kHz to 1 GHz. In intact ribosomes two dispersions were detected: one around 100 kHz and the other one in the MHz region. In separated ribosomal subunits and in ribosomes resuspended in a buffer with no magnesium ions (relaxed ribosomes) only the MHz dispersion was observed. Electrical conductivities of the samples at 1 kHz were also measured. The temperature dependence of the two dispersions was investigated and a tentative attribution was proposed.

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy.

Abstract A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10–90 °C. The Tm of the triplex, 37 °C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the Tm of the triplex was shifted to approximately 32 °C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10–40 °C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 ± 0.5 ns) at 20 °C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 ± 0.2, 12.2 ± 0.2, and 12.0 ± 0.2 Å. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.

γ-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2′-deoxy-7-hydro-8- oxoguanosine lesion

Abstract The influence of the oxidative lesion 2′-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5′-GATCCTCTAGAGTC[G* or G]ACCTGCAGGCATGCA-3′]:[3′-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5′], in which G* is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G*C and G*A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar γ-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G* which are extended for almost one helical turn. Some differences are observed between G*C and G*A duplexes. In particular, in the G*C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G*A sample a shift of the minimum is observed. The hydrodynamic radius Rh derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 ± 0.2 Å between 5 and 40 °C, in all the samples. The torsional constant α of each oligomer decreases when the temperature is raised and the α values of the damaged samples are higher than those of the normal ones.

EFFECT OF THYMINE DIMER INTRODUCTION IN A 21 BASE PAIR OLIGONUCLEOTIDE

Abstract— It is well known that the pyrimidine dimers are the main damage produced by UV radiation on the DNA structure. However, while studies on the photoproduct structure have been carried out extensively, uncertainties still exist on the implication that a single damaging event has on the overall conformation. In particular, the extension of the damage influence on the polynucleotide chain is a matter of debate. This problem is especially important to understanding some steps of the repair mechanisms. In this study we performed a chemical‐physical characterization of 21 base pair oligonucleotides containing a single thymine dimer in one strand. Thermodynamic parameters were determined by means of thermal denaturation experiments, and static fluorescence measurements were performed to unequivocally define the primary structure‐conformation relationship in this specific case. We used hydroxyl radicals, produced by means of γ‐irradiation of the sample solution, to detect fine structure changes. Our data show that the introduction of a single thymine dimer might cause only a slight distortion of the helix geometry, as judged by the evaluation of the enthalpic and the entropic terms and by the small changes observed in the binding of ethidium bromide to DNA. The modifications in the sugar phosphate backbone subsequent to the damaging event are especially evident, near the thymine dimer, toward the 5′‐end direction in the strand containing the dimer.