DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites
Résumé
Tumor suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. The p53 is a nuclear phosphoprotein, and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences differing in their internal symmetry. Binding of p53 to any DNA increased with increasing negative superhelix density (-σ). At -σ<0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNAs bearing both symmetrical and non-symmetrical target sites. On the other hand, at higher -σ target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding due to increasing levels of negative DNA supercoiling. For -σ = 0.07, about a three-fold additional increase of binding was observed for a symmetrical target site, compared to a non-symmetrical target. The p53 target sequences possessing the inverted-repeat symmetry were shown to form cruciform structure in sufficiently negative scDNA. We show that formation of the cruciforms in DNA topoisomers at -σ>0.05 correlates with the extra enhancement of p53-DNA binding.
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