competition assay; p53; DNA sequence-specific binding; supercoiled DNA; cruciform extrusion

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.