DNA BENDING IS ESSENTIAL FOR THE SITE-SPECIFIC RECOGNITION OF DNA RESPONSE ELEMENTS BY THE DNA-BINDING DOMAIN OF THE TUMOR-SUPPRESSOR PROTEIN P53

We have used circular permutation assays to determine the extent and l ocation of the DNA bend induced by the DNA binding domain of human wil d type p53 (p53DBD) upon binding to several naturally occurring DNA re sponse elements. We have found that p53DBD binding induces axial bendi ng in all of the response elements investigated. In particular, respon se elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angl e is similar to 50 degrees), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from similar to 3 7 to similar to 25 degrees). Quantitative electrophoretic mobility shi ft assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the res ponse elements, i.e. the greater the stability of the complex, the mor e the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absenc e of flexible sites in the response elements, may contribute significa ntly to the overall binding affinity of the p53DBD for different seque nces. The results therefore suggest that both the structure and the st ability of the p53-DNA complex may vary with different response elemen ts. This variability may be correlated with variability in p53 functio n.