Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

The tumor suppressor protein p53 is at the nexus of cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds to DNA response elements to regulate transcription of its target genes, a process activated by cellular stress. p53 response elements consist of two decameric half-sites, and most data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding to DNA, unanswered questions remain, due in part to the need for more quantitative and structural studies with the full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding to DNA in real time. The data reveal a dynamic interaction with many p53 binding and dissociation events occurring on single DNA molecules over minutes. We found that p53 tetramers bound to response elements containing only a single half site. The kinetic stability of tetramer/DNA complexes depended on the number of half sites and the helical phasing between them, with the most stable complexes forming on DNA containing two adjacent half sites. The forward rate of binding was not strongly impacted when one half site was mutated. These studies provide real time kinetic measurements of full length p53 tetramers binding to single molecules of DNA, and reveal new insight into the mechanisms by which this nucleoprotein complex forms.