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The DNA Binding Activity of p53 Displays Reaction-Diffusion Kinetics

Peter Hinow ^*, Carl E. Rogers , Christopher E. Barbieri , Jennifer A. Pietenpol , Anne K. Kenworthy  and Emmanuele DiBenedetto ^*

^* Department of Mathematics, Vanderbilt University, Nashville, Tennessee; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee; and Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee

Correspondence: Address reprint requests to Anne K. Kenworthy, Tel.: 615-322-6615; E-mail: anne.kenworthy{at}vanderbilt.edu, or Emmanuele DiBenedetto, Tel.: 615-343-5906; E-mail: em.diben{at}vanderbilt.edu.

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k₁ and k₂, respectively. The diffusion constant of p53-GFP was estimated to be D_p53-GFP = 15.4 µm² s^–1, significantly slower than that of GFP alone, D_GFP = 41.6 µm² s^–1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k₁ = 0.3 s^–1 and k₂ = 0.4 s^–1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.

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