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

Haijun Zhou, Yang Zhang, Zhong Can Ou-Yang, Stuart Lindsay, Xi Z. Feng, Pichumani Balagurumoorthy, Rodney E. Harrington

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

The tumor-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely that these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences based on direct imaging of the topology of microcircles containing the sequences. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microcircles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are markedly puckered with a small excess of negatively writhed molecules. The average measured values of writhe are 0.109 ± 0.013 (for 60 positively writhed molecules) and -0.098 ± 0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in linking number for the positively and negatively writhed molecules prior to ligation, from which we derive a twist mismatch of 178° (overtwist). This is 44.5° for each 42-mer precursor containing a single waf1/cip1/p21 p53 response-element, in good agreement with the range of values deduced by indirect biochemical techniques. The two values of writhe may also be used to determine the ratio 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 that the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These results 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.

Original languageEnglish (US)
Pages (from-to)225-236
Number of pages12
JournalJournal of molecular biology
Volume306
Issue number2
DOIs
StatePublished - Feb 16 2001

Keywords

  • DNA microcircle
  • MacMode atomic force microscopy
  • Waf1 DNA response-element
  • Writhe
  • p53

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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