Effect of p53 protein redox states on binding to supercoiled and linear DNA

The binding of p53 to its DNA consensus sequence is modulated by the redox state of the protein in vitro, We have shown previously that reduced wild-t ype p53 binds strongly to supercoiled DNA (scDNA) regardless of the presenc e or absence of p53CON, Here we compare the effects of oxidation of p53 by azodicarboxylic acid bis-[dimethylamide] (diamide) and other agents on p53 binding to p53CON and to scDNA, Oxidation decreases the binding of p53 to s cDNA; however, under conditions where binding to p53CON in a DNA fragment i s completely abolished, some residual binding to scDNA is still observed. I ncreasing the concentration of oxidized p53 confers minimal changes in p53 binding to both scDNA and p53CON, Reduction of the oxidized protein by dith iothreitol neither restores its binding to DNA nor to p53CON in DNA fragmen ts. In the presence of excess zinc ions, oxidation of p53 is, however, reve rsible. We conclude that the irreversibility of p53 oxidation is due, at le ast in part, to the removal of intrinsic zinc from its position in the DNA binding domain accompanied by a conformational change of the p53 molecule a fter oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein.