Binding of p53 and its core domain to supercoiled DNA

We have compared the binding of human full-length p53 protein (p53; express ed in bacteria and insects) and its isolated core domain (p53CD, amino acid s 94-312; expressed in bacteria) to negatively supercoiled (sc) DNA using g el electrophoresis and immunoblotting. Significant differences were observe d; p53CD produced a relatively small and continuous retardation of scDNA, i n contrast to the ladder of distinct bands formed by p53 in agarose gels. T he ladder produced by full-length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p5 3i). Competition between scDNAs and their linearized (lin) forms showed a p reference for scDNAs by both p53 and p53CD, but the ratios characterizing t he distribution of the protein between sc and lin pBluescript DNAs were sub stantially higher for p53 (sc/lin > 60 in p53b) than for p53CD (sc/lin appr oximate to 4). Strong binding of p53 to scDNA lacking the p53 consensus seq uence may represent a new p53-binding mode, which we tentatively denote sup ercoil-selective (SCS) binding. This binding requires both the C-terminal d omain and the core domain. Targets of this binding may include: (a) DNA seg ments defined both by the nucleotide sequence and local topology and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single-stranded re gions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albe it with reduced affinity due to the absence of contributions from other p53 domains.