CHARACTERIZATION OF BACULOVIRUS RECOMBINANT WILD-TYPE P53 - DIMERIZATION OF P53 IS REQUIRED FOR HIGH-AFFINITY DNA-BINDING AND CYSTEINE OXIDATION INHIBITS P53 DNA-BINDING

A high-yield, rapid and non-denaturing purification protocol for bacul ovirus recombinant wild-type p53 is described. Gel-filtration chromato graphy and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher ol igomers. A gel-mobility-shift assay and protein-DNA cross-linking stud ies demonstrated that purified baculovirus recombinant p53 binds to co nsensus DNA target as a dimer but that additional p53 molecules may th en associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stim ulated sequentially formation of both p53-dimer-DNA and larger p53 -DN A complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the v arious p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues . We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ bindi ng to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-ty pe immunoreactive phenotype of the p53 protein but totally inhibited i ts DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells . The redox status of the nuclear p53 regulates its DNA-binding activi ty in vitro confirming the essential role of the reduced state of cyst eine residues in p53 for detectable DNA-binding activity.