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J. Biol. Chem., Vol. 278, Issue 35, 32587-32595, August 29, 2003

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Analysis of p53 "Latency" and "Activation" by Fluorescence Correlation Spectroscopy

EVIDENCE FOR DIFFERENT MODES OF HIGH AFFINITY DNA BINDING^*

Julian Wölcke , Maurice Reimann , Martin Klumpp , Thomas Göhler , Ella Kim  and Wolfgang Deppert  ¶ ||

From the Evotec OAI, Schnackenburgallee 114, D-22525 Hamburg, Germany, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany, and ^¶Fachbereich Chemie der Universität Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany

The concept that the tumor suppressor p53 is a latent DNA-binding protein that must become activated for sequence-specific DNA binding recently has been challenged, although the "activation" phenomenon has been well established in in vitro DNA binding assays. Using electrophoretic mobility shift assays and fluorescence correlation spectroscopy, we analyzed the binding of "latent" and "activated" p53 to double-stranded DNA oligonucleotides containing or not containing a p53 consensus binding site (DNA_spec or DNA_unspec, respectively). In the absence of competitor DNA, latent p53 bound DNA_spec and DNA_unspec with high affinity in a sequence-independent manner. Activation of p53 by the addition of the C-terminal antibody PAb421 significantly decreased the binding affinity for DNA_unspec and concomitantly increased the binding affinity for DNA_spec. The net result of this dual effect is a significant difference in the affinity of activated p53 for DNA_spec and DNA_unspec, which explains the activation of p53. High affinity nonspecific DNA binding of latent p53 required both the p53 core domain and the p53 C terminus, whereas high affinity sequence-specific DNA binding of activated p53 was mediated by the p53 core domain alone. The data suggest that high affinity nonspecific DNA binding of latent and high affinity sequence-specific binding of activated p53 to double-stranded DNA differ in their requirement for the C terminus and involve different structural features of the core domain. Because high affinity nonspecific DNA binding of latent p53 is restricted to wild type p53, we propose that it relates to its tumor suppressor functions.

Received for publication, April 8, 2003 , and in revised form, June 11, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant De 212/19-4, Deutsche Krebshilfe Grant 10-1417, European Commission Grant QLG1-1999-00273, and by the Fonds der Chemischen Industrie. The Heinrich-Pette-Institut is financially supported by Freie and Hansestadt Hamburg and Bundesministerium für Gesundheit. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 49-40-48051-261; Fax: 49-40-48051-117; E-mail: wolfgang.deppert{at}hpi.uni-hamburg.de.

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