Mendel-Brno 2000

category image Volume: 17
Issue Number 6, Part 2
June 2000

Electron and Scanning Force Microscopy of Interactions of Full Length p53 and its Core Binding Domain with Linear and Supercoiled DNA

We have used transmission electron microscopy (TEM) to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) to linear dsDNA bearing the consensus sequence (p53CON). Both proteins interacted highly specifically and efficiently with the recognition sequence in the presence of 50 mM KCl at low temperature (~4 ºC) while the p53CD also exhibited a strong and specific interaction at physiological temperature. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. Quantitative analysis indicated that the bending induced by p53 varied between ~40 º to 48 º depending on the KCl concentration in the mounting buffer used for sample preparation, while the p53CD bent DNA by 35-37 º independent of salt concentration. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.

The interaction of p53 and p53CD with scDNA molecules was analyzed using both TEM and scanning force microscopy (SFM). The p53CD bound to scDNA bearing the p53CON as a globular complex often located at DNA apices. Binding of the p53CD to scDNA lacking the consensus sequence resulted in the appearance of multiple complexes of various sizes on single DNA molecules. Interaction of full length p53 with the scDNA bearing the p53CON occurred highly specifically at low protein/DNA ratios. Increase in protein concentration resulted in appearance of multiple complexes of various sizes within one DNA molecule. Addition of the p53CD to the scDNA containing a cruciform-forming (AT)34 insert resulted in the binding of the protein exclusively to the cruciform.


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3. Palecek, E., Brazdova, M., Brazda, V., Palecek, J., Subramaniam, V. & Jovin, T.M. Binding of p53 core domain to supercoiled DNA. Submitted

4. Jett, S. D., Cherny, D.I., Subramaniam, V. & Jovin, T.M. Scanning force microscopy of interaction of p53 core domain with supercoiled DNA.(2000) J. Mol. Biol., 299, 587-594.

*present address: Dept. of Cell Biology & Physiology, Univ. of New Mexico,
UNM School of Medicine, 915 Camino de Salud NE,
Albuquerque, NM 8713

D. Cherny, S. Jett*, V.Subramaniam, 1E. Palecek and T. Jovin

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry,
Am Fassberg 11, D-37077 Göttingen, Germany
1Institute of Biophysics, Academy of Sciences of the Czech Republic,
CZ-61265 Brno, Czech Republic