Putrescine, spermidine and spermine are natural polyamines bearing at neutral pH the net electrical charges +2, +3 and +4 respectively. We report here the radioprotective effect of these polyamines on the radiolysis of pBR322 plasmid DNA. We observe a very efficient protection against fast neutron-induced single and double-strand breakage in the presence of spermine and spermidine, and a significantly less efficient protection in the presence of putrescine. An ionic strength dependence is observed for spermidine and spermine, but not for putrescine. Circular dichroism measurements show spermidine- and spermine-induced structural modifications of DNA, i.e. the formation of tightly packaged condensates in the concentration range corresponding to radioprotection. No structural change is observed for concentrations of putrescine affording radioprotection. We explain the radioprotection by: (1) the scavenging of OH radicals in the bulk, essentially observed in the case of putrescine; (2) a local scavenging at the sites of binding of polyamines; and (3) the reduced accessibility of the attack sites in the condensed structures induced by spermine or spermidine.

Ionizing radiations induce numerous damages in DNA, especially strand breaks. The hydroxyl radical OH., produced by the radiolysis of water, is mainly responsible for this effect. The fact that strand breakage occurs at all nucleotides and that bound proteins may locally radioprotect DNA at the binding site lead us to develop a radiolytic footprinting method to study DNA-protein interactions. Three different radiations were used: beta rays, gamma photons, and fast neutrons. In order to validate this technique, three well-known interaction systems were tested: the lac repressor-lac operator of Escherichia coli, the cyclic AMP receptor protein (CRP) of E. coli and its specific site in the lac regulation region, and the core nucleosome. Radiolytic footprinting gives results similar to those obtained by more classical probes: DNase I, complexes of orthophenanthroline (OP) and copper, complexes of ethylenediaminetetraacetate ion (EDTA) and iron, and UV light. For the same system (lac repressor), irradiation with either gamma photons or fast neutrons gives identical results.

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Abstract— The photochemical reactions of benzophenone and acetophenone with purine and pyrimidine derivatives in aqueous solutions have been investigated by flash photolysis and steady‐state experiments. Upon excitation of these two ketones in aqueous solutions, two transient species are observed: molecules in their triplet state and ketyl radicals. The triplet state lifetimes are 65 μsec for benzophenone and 125 μsec for acetophenone. The ketyl radicals disappear by a second order reaction, controlled by diffusion. In the presence of pyrimidine derivatives, the triplet state is quenched and the ketyl radical concentration is decreased without any change in its kinetics of disappearance. Ketone molecules in their triplet state react with purine derivatives leading to an increase in the yield of ketyl radicals due to H‐atom abstraction from the purines. Steady‐state experiments show that benzophenone and acetophenone irradiated in aqueous solution at wavelengths longer than 290 nm undergo photochemical reactions. The rate of these photochemical reactions is increased in the presence of pyrimidine derivatives and even more in the presence of purine derivatives. Following energy transfer from the triplet state of benzophenone to diketopyrimidines, cyclobutane dimers are formed. The energy transfer rate decreases in the order orotic acid > thymine > uracil. Benzophenone molecules in their triplet state can also react chemically with pyrimidine derivatives to give addition photoproducts. All these results are discussed with respect to photosensitized reactions in nucleic acids involving ketones as sensitizers.

We are presently investigating the origin and the nature of molecular interactions responsible for the specific recognition of nucleic acid sequences by proteins (see refs. 1-4 for previous publications). Since we are interested in the role played by aromatic amino acids, we have studied the binding of oligopeptides containing lysyl and tryptophyl residues such as Lys-Trp-Lys to different nucleic acids and polynucleotides. Proton magnetic resonance (2, 4) and circular dichroism* studies have provided evidence for a stacking interaction of the tryptophyl ring with bases. A similar conclusion has also been reached by Gabbay and coworkers for different tryptophan-containing peptides (5, 6). Fluorescence and phosphorescence studies at low temperature have shown that the tryptophyl ring stacked with bases acts as a trap for the triplet excitation energy (7). At room temperature, fluorescence spectroscopy is appropriate for study of the binding of tryptophyl-containing peptides to nucleic acids since only the tryptophyl ring emits fluorescence in fluid medium. The weak fluorescence of nucleic acid bases (8, 9) at room temperature has a quantum yield that is 3 orders of magnitude smaller than that of tryptophan.A quantitative analysis of fluorescence data is presented here which shows that two types of complexes are formed when oligopeptides such as Lys-Trp-Lys bind to nucleic acids. The role of the local structure of nucleic acids in the binding process is investigated by comparing the behavior of heatdenatured or UV-irradiated DNA with native double-stranded DNA with respect to stacking of the tryptophyl ring with bases. It is also shown that the tryptophyl ring is able to photosensitize the splitting of thymine dimers in UV-irradiated DNA. EXPERIMENTALFluorescence measurements were made with a Jobin-Yvon spectrofluorimeter that was modified to correct for lamp fluctuations by deflecting part of the incident beam onto a rhodamine B quantum counter. The sample was contained in a 5-mm quartz Suprasil thermostated cell (usually 2°).Difference absorption spectrophotometry measurements have shown that the binding of tryptophan-containing peptides to nucleic acids is accompanied by a small but reproducible absorption change. The excitation wavelength was therefore chosen at the isosbestic point of these difference spectra (292 nm).To correct fluorescence intensities for the screening effect of the nucleic acid at the excitation wavelength, peptidenucleic acid complexes were dissociated by increasing the ionic strength (2, 12). At high ionic strength (0.5 M NaCl), only the screening effect of the nucleic acid was assumed to lead to an apparent decrease of the fluorescence quantum yield of the peptide. All fluorescence quantum yields were measured with respect to that of the free peptide and corrected for the screening effect of the nucleic acid.Polynucleotides and oligopeptides were purchased from RESULTSThe binding of peptides containing lysyl and tryptophyl residues to nucleic acids is accompanied by a decrease o...