Michel Charlier

DNA Radiolysis by Fast Neutrons

The effects of fast neutron irradiation on DNA were studied using DNA of the pBR322 plasmid (4362 base pairs), and the results compared to those obtained with 60Co gamma rays. Irradiation of the plasmid DNA in solution with a neutrons beam (p34+Be) of the CERI (CNRS Orléans) cyclotron (with a flat energy spectrum from 34 MeV to low energies) results in half the yield of single-strand breaks (ssb), and 1.5 times higher yield of double-strand breaks (dsb) for neutrons as compared to gamma-rays. Possible specificity of the neutron-induced breaks was examined: the scavenging of OH. radicals by 0.1 mol dm-3 ethanol inhibits all neutron-induced ssb, but only 85 per cent of the dsb. For gamma-irradiation, both ssb and dsb are completely inhibited in these conditions. These results suggest at least three different origins for neutron-induced dsb. The occurrence of around 30 per cent of dsb can be explained by a radical transfer mechanism (proposed by Siddiqi and Bothe (1987) for gamma-irradiation). Around 55 per cent of dsb may be due to the non-random distribution of radicals in high-density tracks of the secondary particles of neutrons, which results in a simultaneous attack of the two strands by OH. radicals. These first two processes are both OH.-mediated and thus are sensitive to ethanol. The direct effect of fast neutrons and their secondaries (recoil protons, alpha-particles and recoil nuclei) can account for the remaining 15 per cent of dsb, not inhibited by 0.1 mol dm-3 ethanol.

Radioprotection of DNA by Polyamines

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.

PHOTOCHEMICAL REACTIONS OF AROMATIC KETONES WITH NUCLEIC ACIDS AND THEIR COMPONENTS I—. PURINE AND PYRIMIDINE BASES AND NUCLEOSIDES.*

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.

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS BY INDOLE DERIVATIVES AND BY TRYPTOPHAN-CONTAINING OLIGOPEPTIDES AND PROTEINS

Abstract— Indole derivatives including tryptophan can be used as photosensitizers of the splitting of pyrimidine dimers. The reaction can take place in frozen aqueous solutions as well as in fluid medium. Electron transfer from the indole ring to the dimer appears to be involved in the photosensitized reaction. Solvated electrons produced by flash photolysis in the presence of indoles or by pulse radiolysis are also able to split thymine dimers.

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS IN DNA BY INDOLE DERIVATIVES AND TRYPTOPHAN-CONTAINING PEPTIDES

Abstract—Indole derivatives, such as serotonin or the oligopeptide Lys‐Trp‐Lys, are able to photosensitize the splitting of thymine dimers in DNA. These indole derivatives have to be bound to DNA in order to efficiently photosensitize the splitting reaction. Serotonin may also induce the photosensitized formation of thymine‐containing dimers in native DNA. In this case, an equilibrium is reached when 5 per cent of the total thymines are dimerized. In both cases (splitting and dimer formation), the formation of electron donor‐acceptor complexes between either dimers or two adjacent thymine monomers, and excited indole rings, could be an intermediate step in the reactions. Thymine‐dimer splitting would then result from an electron transfer reaction involving the indole ring as the electron donor. These results are discussed with respect to the mechanism of action of the photoreactivating enzyme.

Photosensitized splitting of thymine dimers in DNA by gene 32 protein from phage T 4.

Abstract The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA.

Radioprotection of DNA by spermine: a molecular modelling approach

PURPOSE To observe and explain the sequence-dependence of DNA radioprotection by spermine. MATERIALS AND METHODS Sequencing gel electrophoresis was used to analyse the probability of frank strand break (FSB) induction at each nucleotide site. Molecular modelling of complexes of DNA with spermine molecules and of a curved electrically null DNA has been performed. RESULTS The effect of spermine on radiation-induced strand breakage varied significantly along the studied fragment. At low spermine concentration, some sequences were protected while others were unprotected. Molecular modelling calculations show that the most electro-negative sites are located in the minor or in the major groove of DNA. The positively charged spermine (Z=+4) should preferentially bind to such sites. When bound in the minor groove, spermine triggers a reduction of the accessibility of radiolytic attack sites to OH* radicals. This is due to induced structural modifications and to the masking of attack sites. In the case of major groove binding, no reduction of accessibility occurs. This type of binding can explain the lack of protection of sequences with electro-negative sites in the major groove. At high spermine concentration, the fragment is strongly protected. A nucleosome-like pattern of breakage with periodically distributed regions of protection was observed. Molecular modelling calculations show that the accessibility of the attack sites in a curved electrically null DNA is also periodically reduced. CONCLUSIONS Molecular modelling of DNA-spermine complexes that takes into account the electrostatic properties of DNA, allows an explanation of the experimentally observed effects of spermine on DNA radiosensitivity.

Metal Ions Protect DNA Against Strand Breakage Induced by Fast Neutrons

Single and double strand breaks (SSB and DSB) are induced by fast neutrons in plasmid (pBR322) DNA in 1 mM potassium phosphate buffer (pH 7.25). Increasing the concentration of monovalent (Na+, Cs+, Li+), divalent (Mg2+, Ca2+) and trivalent (Al3+, Co3+ (NH3)6) metal cations strongly decreases the yield of DSB. The extent of the observed protection depends on the valence of the cation. The production of SSB is only slightly decreased, except for Al3+ and Co3+ (NH3)6, whose effects are particularly large (complete protection at 1 and 0.1 mM respectively). Circular dichroism spectra show that Al3+ induces an important structural change of DNA at the ion concentration where the protection becomes total. This change is probably a condensation (collapse), as in the well-known case of Co3+ (NH3)6. Our results suggest two mechanisms of protection by metal ions: (i) the induction of structural changes of DNA, that render less accessible the critical sites of attack by OH. radicals; and (ii) the stabilization of the double helical regions between two close-set nicks on opposite strands, that hinders the effective double strand breakage of DNA.

Photosensitized splitting of pyrimidine dimers by indole derivatives

Abstract Excitation of tryptophan and 5-hydroxytryptophan at wavelengths longer than 290 and 310 nm, respectively, in the presence of pyrimidine dimers leads to a sensitized splitting of these dimers. The reaction is more efficient in frozen than in fluid aqueous solutions. The fluorescence of tryptophan and 5-hydroxytryptophan is quenched by thymine dimers. These results suggest that splitting occurs as a result of electron transfer from the excited indole derivative to the pyrimidine dimer.

RADACK, a stochastic simulation of hydroxyl radical attack to DNA.

Abstract RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes. It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling. The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively. The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed. The calculated relative probabilities of OH. radical attack to all reaction sites are compared to experimental FSB and ARB values. By a fitting procedure, the relative efficiencies of conversion of the C4′ and C5′-centred radicals into FSB, ϵ (C4′): ϵ (C5′), and the relative efficiencies of base radicals—to—ARB conversion, ϵ (T): ϵ(A): ϵ(C): ϵ(G), are then deduced for each DNA form. The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems: the lac repressor-lac operator complex and the nucleosome core.