ANALYSIS OF P53 QUATERNARY STRUCTURE IN RELATION TO SEQUENCE-SPECIFICDNA-BINDING

Quaternary interactions of p53 influence its tertiary structure which, in turn, is critical for sequence-specific DNA binding and tumour sup pressor function. Given its regulatory potential we have sought to def ine the quaternary structure of p53 involved in sequence-specific DNA binding. Double stranded DNA [5'-GGACATGCCCG GGCATGTCC-3'; Funk et al. (1992) Mol. Cell. Biol., 12, 2866-2871] was used to test p53 binding capacity in vitro. The p53 protein was translated in vitro and size fr actionated prior to the DNA binding reaction. Two independent DNA bind ing assays were employed. The first detected electromobility shift of P-32-labelled DNA and was carried out in the presence of PAb421, which stabilises and supershifts p53-DNA complexes. The second detected S-3 5-labelled p53 bound to biotinylated target DNA in the absence of PAb4 21. Sequence-specific DNA binding was found to be a property of full l ength, oligomeric p53. Greatest binding activity involved tetramers an d/or higher molecular weight forms of p53, minimal binding was observe d for dimers. This size profile was unaffected by PAb421 and it theref ore seems unlikely that PAb421 dissociates high molecular weight forms of p53 into dimers. We conclude that high molecular weight forms of p 53 are the most effective structures for sequence-specific DNA binding in vitro; these structures may represent tetramers and/or heterogeneo us complexes of p53 with other proteins.