Different regulation of the p53 core domain activities 3 '-to-5 ' exonuclease and sequence-specific DNA binding

In this study we further characterized the 3'-5' exonuclease activity intri nsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-termi nal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activit y by at least 10-fold, indicating that this activity, like sequence-specifi c DNA binding, is negatively regulated by the C-terminal basic regulatory d omain of p53. However, treatments which activated sequence-specific DNA bin ding of p53, like binding of the monoclonal antibody PAb421, which recogniz es a C-terminal epitope on p53, or a higher phosphorylation status, strongl y inhibited the p53 exonuclease activity. This suggests that at least on fu ll-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Followi ng up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombi nation intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such subst rates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to th e binding assay indeed started the p53 exonuclease and promoted rapid degra dation of the bound, but not of the unbound, substrate, indicating that spe cifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.