Conformation and rigidity of DNA microcircles containing waf1 response element for p53 regulatory protein

The tuner-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely tha t these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences b ased on direct imaging of the topology of microcircles containing the seque nces. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microci rcles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are m arkedly puckered with a small excess of negatively writhed molecules. The a verage measured values of writhe are 0.109 +/- 0.013 (for 60 positively wri thed molecules) and -0.098 +/- 0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in Linking number for the posit ively and negatively writhed molecules prior to ligation, from which we der ive a twist mismatch of 178 degrees (overtwist). This is 44.5 degrees for e ach 42-mer precursor containing a single waf1/cip1/p21 p53 response-element , in good agreement with the range of values deduced by indirect biochemica l techniques. The two values of writhe may also be used to determine the ra tio of the bending (B) to twisting (C) rigidity, yielding B/C = 0.23. This is about one-third of the value for long, random-sequence DNA, suggesting t hat the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These result s support the idea, proposed by us earlier, that torsional stress may play a role in the regulation of p53 binding through modulation of twist at the binding site. (C) 2001 Academic Press.