Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno

References provided by Crossref.org

An upward 9.4 T static magnetic field inhibits DNA synthesis and increases ROS-P53 to suppress lung cancer growth

The Rich World of p53 DNA Binding Targets: The Role of DNA Structure

p73, like its p53 homolog, shows preference for inverted repeats forming cruciforms

Recognition of Local DNA Structures by p53 Protein

p53 Specifically Binds Triplex DNA In Vitro and in Cells

Wild-type p53 binds to MYC promoter G-quadruplex

Differential salt-induced dissociation of the p53 protein complexes with circular and linear plasmid DNA substrates suggest involvement of a sliding mechanism

DNA and RNA quadruplex-binding proteins

Preferential binding of hot spot mutant p53 proteins to supercoiled DNA in vitro and in cells

Cruciform structures are a common DNA feature important for regulating biological processes

Activation of the DNA-binding ability of latent p53 protein by protein kinase C is abolished by protein kinase CK2

Role of tumor suppressor p53 domains in selective binding to supercoiled DNA