Cooperative binding of tetrameric p53 to DNA.
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.
Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, United Kingdom.
Richard L
RL
Weinberg
Dmitry B
DB
Veprintsev
Alan R
AR
Fersht
D001483:Base Sequence; D002021:Buffers; D004247:DNA / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D046911:Macromolecular Substances; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
D002021:Buffers; D046911:Macromolecular Substances; D021122:Protein Subunits; D016159:Tumor Suppressor Protein p53; D004247:DNA