Effects of oxidation agents and metal ions on binding of p53 to supercoiled DNA

Wild type human full length (f.l.) tumor suppressor p53 protein binds prefe rentially to super coiled (sc) DNA in vitro both in the presence and absenc e of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with a ntibody DO-1 corresponded to the ethidium stained retarded bands. The inten sity and the number of bands of p53-scDNA complex were decreased by physiol ogical concentrations of unchelated zinc ions. Nickel and cobalt ions inhib ited binding of p53 to scDNA and to p53CON in linear DNA fragments less eff iciently than zinc. Compared to the intrinsic zinc strongly bound to Cys 17 6, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of addition al Zn2+ to p53 was much weaker as shown by an easy removal of the latter io ns by low concentrations of EDTA. Oxidation of the protein with diamide res ulted in a decrease of the number of the retarded bands. Under the same con ditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.l. p53 with diami de was irreversible and was not reverted by an excess of DTT. We showed tha t in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such e ffect. We suggested that the irreversibility of p53 oxidation was due, at l east in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc i on is coordinated in the reduced protein) accompanied by a change in the p5 3 conformation. Binding of C-terminal anti-p53 antibody also protected bact erially expressed protein against irreversible loss of activity due to diam ide oxidation. Binding the human p53 core domain (segment 94-312) to scDNA greatly differed from that observed with the full-length p53. The core doma in did not posses the ability to bind strongly to many sites in scDNA regar dless of the presence or absence of p53CON suggesting involvement of some o ther domain (probably C-terminal) in binding of the full-length p53 to scDN A. Supershift experiments using antibodies against p53 N- or C-terminus sug gested that in oxidized p53, scDNA binding through the C-terminus gained im portance.