M. Spotheim-Maurizot

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.

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.

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.

Radiation Disrupts Protein–DNA Complexes through Damage to the Protein. The lac Repressor–Operator System

Abstract Eon, S., Culard, F., Sy, D., Charlier, M. and Spotheim-Maurizot, M. Radiation Disrupts Protein–DNA Complexes through Damage to the Protein. The lac Repressor–Operator System. Radiat. Res. 156, 110–117 (2001). Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein–DNA recognition, the entire regulation process may be perturbed. We have studied the effect of γ rays on a model regulatory system, the E. coli lactose repressor–operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, but not by the addition of nonirradiated DNA. Thus radiation disrupts the DNA–protein complex by affecting the binding ability of the protein. This interpretation is supported by the dramatic loss of binding ability of a free irradiated repressor toward nonirradiated DNA. Interestingly, the dose necessary for the disruption of the irradiated complex is higher than that for inducing the complete loss of the binding ability of the free irradiated repressor. This may be due to the protection of key amino acids by the bound DNA. As seen from calculations of the accessibility of amino acids to radiolytic OH·, the protection is due to both masking and conformational effects.

Radiation damage to DNA in DNA-protein complexes.

The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals.

In Vitro Studies with Methylproamine A Potent New Radioprotector

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is ∼100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)2 confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.

Radiation Affects Binding of Fpg Repair Protein to an Abasic Site Containing DNA

Abstract Gillard, N., Begusova, M., Castaing, B. and Spotheim-Maurizot, M. Radiation Affects Binding of Fpg Repair Protein to an Abasic Site Containing DNA. Radiat. Res. 162, 566– 571 (2004). During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA. Thus irradiation hinders Fpg-DNA binding because of the damage to the protein. Using our radiolytic attack simulation procedure RADACK (Begusova et al., J. Biomol. Struct. Dyn. 19, 141–157, 2001), we reveal the potential hot spots for damage in the irradiated protein. Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg. The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately. As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.