Analysis of p53 quaternary structure in relation to sequence-specific DNA binding

Abstract

Quaternary interactions of p53 influence its tertiary structure which, in turn, is critical for sequence-specific DNA binding and tumour suppressor function. Given its regulatory potential we have sought to define the quaternary structure of p53 involved in sequence-specific DNA binding. Double stranded DNA [5'-GGACATGCCCGGGCATGTCC-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 fractionated prior to the DNA binding reaction. Two independent DNA binding assays were employed. The first detected electromobility shift of 32P-labelled DNA and was carried out in the presence of PAb421, which stabilises and supershifts p53-DNA complexes. The second detected 35S-labelled p53 bound to biotinylated target DNA in the absence of PAb421. Sequence-specific DNA binding was found to be a property of full length, oligomeric p53. Greatest binding activity involved tetramers and/or higher molecular weight forms of p53, minimal binding was observed for dimers. This size profile was unaffected by PAb421 and it therefore seems unlikely that PAb421 dissociates high molecular weight forms of p53 into dimers. We conclude that high molecular weight forms of p53 are the most effective structures for sequence-specific DNA binding in vitro; these structures may represent tetramers and/or heterogeneous complexes of p53 with other proteins.