title: Enhancement of p53 sequence-specific binding by DNA supercoiling.
abstract: Using a new competition assay, we investigated the effect of DNA negative supercoiling on the DNA sequence-specific binding (SSDB) of human wild-type (wt) p53 protein. We found that supercoiled (sc) pBluescript DNAs with different inserted p53 target sequences were stronger competitors than a mixture of scDNA pBluescript with the given 20-mer target oligodeoxynucleotide. ScDNAs were always better competitors than their linearized or relaxed forms. Two DNAs with extruded cruciforms within the target sequence were the best competitors; removal of the cruciforms resulted in a decrease of competitor strength. In contrast to the full-length wt p53, the deletion mutant p53CDelta30 and the p53 core domain (93-312 aa) showed no enhancement of p53 SSDB to scDNA, suggesting that, in addition to the p53 core domain, the C-terminal was involved in this binding. We conclude that cruciforms and DNA bends contribute to the enhancement of p53 SSDB to scDNA and that the DNA supercoiling is an important determinant in the p53 sequence-specific binding. Supercoiling may thus play a significant role in the complex p53-regulatory network.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno 612 65, Czech Republic. palecek@ibp.cz
authors.forename: Emil
authors.initials: E
authors.lastname: Palecek
authors.forename: Václav
authors.initials: V
authors.lastname: Brázda
authors.forename: Eva
authors.initials: E
authors.lastname: Jagelská
authors.forename: Petr
authors.initials: P
authors.lastname: Pecinka
authors.forename: Lenka
authors.initials: L
authors.lastname: Karlovská
authors.forename: Marie
authors.initials: M
authors.lastname: Brázdová
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D016384:Consensus Sequence; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004278:DNA, Superhelical; D016159:Tumor Suppressor Protein p53