L'attaque radiolytique de l'ADN dans les complexes acides nucléiques - protéines

Une étude physico-chlmlque et moléculaire de la radiolyse de l'ADN en interaction avec des protéines, montre que les protéines fixées sur TADN protègent très efficacement Ieur site de fixation contre l'agression des radicaux radioinduits dans l’eau, par masquage physique. Les résultats observés ne dépendent pas de la qualité du rayonnement (rayons β, γ et neutrons rapides). Cela nous a permis de mettre au point une méthode d'empreinte moléculaire par radiolyse, utilisable in vivo

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 γ-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