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

Cherny, D. I., Striker, G., Subramaniam, V., Jett, S. D., Palecek, E., & Jovin, T. M. (1999). DNA bending due to specific p53- and p53 core domain-DNA interactions visualized by electron microscopy. Journal of Molecular Biology, 294, 1015-1026.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-FA54-5 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-E856-6
Genre: Journal Article

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 Creators:
Cherny, D. I.1, Author              
Striker, G.1, Author              
Subramaniam, V.1, Author              
Jett, S. D.1, Author              
Palecek, E., Author
Jovin, T. M.1, Author              
Affiliations:
1Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              

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Free keywords: Scanning force microscopy; Suppressor protein p53; p53; Binding domain; Oligomerization domain; Nucleic acids; Site; Complexes; Recognition; Polymerase; Peptides
 Abstract: We have used transmission electron microscopy 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) encoding amino acid residues 94-312, to linear double-stranded DNA bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCI at low temperature (similar to 4 degrees C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. 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 induced by p53 varied from similar to 40 degrees to 48 degrees upon decreasing of the KCI concentration from 50 mM to similar to 1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35-37 degrees for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (sigma approximate to 39 degrees) than at high salt concentration (sigma approximate to 31 degrees) or for p53CD (sigma approximate to 24-27 degrees). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.

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Language(s): eng - English
 Dates: 2005-07-081999
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: eDoc: 224259
Other: 11467
 Degree: -

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Title: Journal of Molecular Biology
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 294 Sequence Number: - Start / End Page: 1015 - 1026 Identifier: -