Analysis of p53 "latency" and "activation" by fluorescence correlation spectroscopy. Evidence for different modes of high affinity DNA binding.
The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNAspec or DNAunspec, respectively). In the absence of competitor DNA, latent p53 bound DNAspec and DNAunspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNAunspec and concomitantly increased the binding affinity for DNAspec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNAspec and DNAunspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.
Wölcke
Julian
J
Evotec OAI, Schnackenburgallee 114, D-22525 Hamburg, Germany.
Reimann
Maurice
M
Klumpp
Martin
M
Göhler
Thomas
T
Kim
Ella
E
Deppert
Wolfgang
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009841:Oligonucleotides; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA