Book of Abstracts: Albany 2007
June 19-23 2007
Mutant p53 Interactions with Supercoiled DNA
DNA topology, supercoiled structure of DNA, plays an important role in the regulation of various cellular events via specific and non-specific DNA interactions with proteins. Usually natural negative DNA supercoiling modulates these interactions and hence the efficiency/specificity/stability of the protein-DNA complexes.
Two DNA-binding domains (core domain and C-terminal) of full length wild-type (wt) p53 protein are able to recognize several specific DNA motifs (e.g., specific consensus sequence, ssDNA, structural DNA motifs) (1) as well as supercoiled DNA (scDNA). Full length wtp53 binds selectively to scDNA regardless of the presence or absence of the p53 consensus sequence (p53CON) (2). Earlier we showed that p53 sequence-specific DNA binding can be enhanced by negative supercoiling. The extent of the enhancement depends on the p53 target sequence (3). In competition experiments with linear (lin) DNA, full length p53 binds scDNA with a high preference (supercoil-selective, SCS binding). Electron microscopy revealed formation of nucleoprotein filaments at higher protein/DNA ratios. It has been proposed that the SCS DNA binding involves cooperative interactions of the oligomeric p53 C-terminal domain with two segments of the DNA double helix within the plectonemic DNA superhelix, and stabilization of the complexes (filaments) by further protein?DNA and protein-protein interactions (4).
We studied bacterially expressed full-length mutant p53 proteins (mutp53), including most of the ?hot spot? mutants (R175H, G245S, R248W, R249Q, R273C, R273H) by electrophoretic and immunoprecipitation techniques. Mutp53 has been reported to be unable of recognizing p53CON sequences in short linear DNA, but to bind selectively to oligonucleotides in stem-loop forms (5). We found that some of mutp53 proteins recognized also p53CON in longer DNA (∼500bp; observed at 0°C). Moreover, they retained ability to interact with natural supercoiled DNA regardless of the presence of p53CON. We studied SCS DNA binding of mutp53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the C-terminal domain). Using p53 deletion mutants, we have shown that the p53 C-terminal DNA binding site is critical for this binding. These data indicate that the C-terminus is primarily responsible for the p53 SCS DNA binding.
Considering the repeatedly proposed active role of the mutant p53 in tumorgenesis these observations may provide important insights into the molecular mechanisms that underlie the mutant p53 gain of function.
This work was supported by QLGA-CT-2001-52001(EC), MERG-6-CT-2005-014875 (EC), 1K04119 (Ministry of Education, Youth and Sports), 204/06/P369 (Grant Agency of Science of CR) to M.B, Research Centre No. LC06035.
References and Footnotes
Institute of Biophysics Academy of Sciences CR