Radiation affects binding of Fpg repair protein to an abasic site containing DNA

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

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

Radiolytic signature of Z-DNA.

Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of B- and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARB) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARB. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3' cytosine is more important in Z-DNA than in B-DNA