Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation

Genotoxic stress activation of the tumor suppressor transcription factor p5 3 involves post-translational C-terminal modifications that increase both p rotein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated g enes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encodin g a ubiquitin ligase that targets p53 for proteolytic degradation), The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 bindi ng consensus sequence, while the p53 binding site in the Mdm2 promoter cont ains two consensus sequences linked by a 17 bp spacer, Binding of recombina nt p53 protein to the p21/WAF1 binding site required monoclonal antibody PA b421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each co nsensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consen sus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer reg ion from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm 2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady in duction of Mdm2 protein would maintain p53 protein at low levels until post -translational modifications following DNA damage increased p53 activity to wards other genes, mediating p53 growth inhibitory and apoptotic activities .