DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy

We have used transmission electron microscopy to analyze the specificity an d the extent of DNA bending upon binding of full-length wild-type human tum or suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding am ino acid residues 94-312, to linear double-stranded DNA bearing the consens us sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted w ith high specificity and efficiency with the recognition sequence in the pr esence of 50 mM KCI at low temperature (similar to 4 degrees C) while the p 53CD also exhibits a strong and specific interaction at physiological tempe rature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induc ed by p53 varied from similar to 40 degrees to 48 degrees upon decreasing o f the KCI concentration from 50 mM to similar to 1 mM in the mounting buffe r used for adsorption of the complexes to the carbon film surface. The p53C D bent DNA by 35-37 degrees for all salt concentrations used in the mountin g buffer. The bending angle of the p53/DNA complex under low salt condition s showed a somewhat broader distribution (sigma approximate to 39 degrees) than at high salt concentration (sigma approximate to 31 degrees) or for p5 3CD (sigma approximate to 24-27 degrees). Together, these results demonstra te that the p53CD has a dominant role in complex formation and that the com plexes formed both by p53 and p53CD under moderate salt conditions are simi lar. However, the dependence of the bending parameters on ambient condition s suggest that the segments flanking the p53CD contribute to complex format ion as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed. (C) 1999 Academic Press.