TUMOR-SUPPRESSOR PROTEIN P53 BINDS PREFERENTIALLY TO SUPERCOILED DNA

Wild type human tumor suppressor protein p53 (expressed in insect cell s) binds strongly to negatively supercoiled (sc) plasmid DNA at a nati ve 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 consens us 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 n umber, In DNA samples partially relaxed by topoisomerase I p53 binds p referentially to the scDNA molecules with the largest negative superhe lix 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 o f p53 for negatively supercoiled DNA is greater than that for the cons ensus sequence in linear fragments, However, thermally denatured linea rized 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 c onsensus sequence, Thus, it appears that interactions involving both t he core domain and the C-terminal domain regulate the binding of p53 t o scDNA, The above results are not restricted to human p53; the wild t ype rat p53 protein also results in the retardation of scDNA on agaros e gels, The biological implications of the novel DNA binding activitie s of p53 are discussed.