Cooperative binding of tetrameric p53 to DNA

J Mol Biol. 2004 Aug 27;341(5):1145-59. doi: 10.1016/j.jmb.2004.06.071.


We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Buffers
  • DNA / metabolism*
  • Fluorescence Polarization
  • Humans
  • Macromolecular Substances
  • Protein Binding
  • Protein Structure, Quaternary*
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Tumor Suppressor Protein p53 / chemistry*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Ultracentrifugation


  • Buffers
  • Macromolecular Substances
  • Protein Subunits
  • Tumor Suppressor Protein p53
  • DNA