4 P53 DNA-BINDING DOMAIN PEPTIDES BIND NATURAL P53-RESPONSE ELEMENTS AND BEND THE DNA

Recent structural studies of the minimal core DNA-binding domain of p5 3 (p53DBD) complexed to a single consensus pentamer sequence and of th e isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered, To better understand these relations hips, we have examined the binding of the p53DBD to two biologically i mportant full-response elements (the WAF1 and ribosomal gene cluster s ites) by using DNA circularization and analytical ultracentrifugation, We show that the p53DBD binds DNA strongly and cooperatively with p53 DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the m ean apparent K-d is (8.3 +/- 1.4) x 10(-8) M, and no intermediate spec ies of lower stoichiometries can be detected, We show further that com plex formation induces an axial bend of at least 60 degrees in both re sponse elements. These results, taken collectively, demonstrate that p 53DBD possesses the ability to direct the formation of a tight nucleop rotein complex having the same 4:1 DNA-binding stoichiometry as wild-t ype p53 which is accompanied by a substantial conformational change in the response-element DNA, This suggests that the p53DBD may play a ro le in the tetramerization function of p53, A possible role in this reg ard is proposed.