SUNY at Albany
June 19-23, 2001
Binding of p53 Protein to Double-Stranded DNA
In order to better characterize the interactions of p53 protein with dsDNA we analyzed the binding of various forms of p53 protein (full length purified from baculovirus and bacteria, core binding domain, truncated form of the full length lacking 30 aa at the C-terminal) with dsDNA of different composition and conformation (pBluescript DNA or pBluescript DNA with a cloned recognition sequence, both in linear or positively and negatively supercoiled forms) by means of electron microscopy.
We have found that each form of the protein interacts with the recognition p53 sequence within linear DNA. However, in addition to sequence-specific binding all forms interact with other sequences, thus forming stable complexes albeit with lesser efficiency. Mapping of the additional binding sites showed that they are not distributed randomly over the entire sequence of pBluescript DNA and probably represent degenerative forms of the consensus sequence.
In most cases bound proteins were imaged as individual globular structures of similar size (for each given protein). However, bound protein exhibited a degree of cooperativity, mediated by protein-protein interactions, thus leading to DNA looping. Furthermore, protein binding led to the formation protein-DNA filaments in which two DNA strands are stabilized via bound protein molecules. The latter effects were most pronounced for the bacterially expressed full length p53. Analysis of these complexes showed that they were positioned at the sites of preferential binding to DNA.
In many respects, all forms of p53 interact with scDNA (5-7 positive or negative superhelical turns) in a similar way as with their linear counterparts. However, the interaction with scDNA is more efficient relative to linear molecules irrespective to the sign of superhelicity. Secondly, the interaction is not affected by DNA composition. More than one binding complex, mostly of globular shape, was observed with a given DNA molecule. Due to the inherent topology of scDNA the cooperative binding of p53 was also enhanced, leading to the formation of the complexes apparently at DNA crossovers and ultimately to protein-DNA filaments. The later effect was most prominent for p53 purified from bacteria, irrespective of the sign of superhelicity and DNA origin (i.e. with or without the recognition sequence).
We hypothesize that significant diversity of the recognition sequences together with DNA-protein and protein-protein interactions lead to a variety of imaged p53-DNA complexes modulated by the protein form and DNA topology. The latter contribution being of central significance. The interaction of p53 with DNA has also been studied by dynamic scanning force microscopy.
References and Footnotes
Dmitry I. Cherny (1), Marie Brazdova (2), Jan Palecek (2), Emil Palecek (2) and Thomas M. Jovin (1)
(1)Dep't./Mol. Bio., Max Planck Inst./Biophys. Chem., Am Fassberg 11, D-37077 Göttingen, Germany (2)Inst./Biophy., Acad./Science/Czech Republic, Brno, Czech Republic email: email@example.com Ph: (+)49 551 201 1383; F: (+)49 551 201 1467