Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Binding of Latent and Activated Protein p53 to Supercoiled and Chemically Damaged DNA

Tumor suppressor protein p53 is a DNA-binding protein possessing two DNA binding sites (1-4 and references therein). The p53 core domain is responsible for p53 sequence-specific DNA binding as well as non-specific binding to long single- or double-stranded DNA molecules; it is also essential for p53 binding to DNA recombination intermediates (1). The other DNA-binding site that is situated at the protein C-terminus is known to bind damaged DNA. The latter site coincides with a negative-regulating region which inhibits DNA binding functions in the post-translationally unmodified (latent) form of the protein (4). Using electrophoretic mobility shift assay in agarose gels and immunoblotting analysis we studied binding of latent and activated p53 to negatively supercoiled (sc) and linearized (lin) plasmid DNA, and to cisplatin-modified DNA fragments. Latent form of p53 exhibited highly preferential binding to scDNA not containing the p53 consensus sequence (p53CON) (2,3) and tight binding to platinated but not unmodified linDNA. Activated p53 significantly bound sc, lin and platinated DNA, without any substantial preference. This binding was strongly decreased upon oxidation of the protein cysteine residues while binding of the latent protein to sc and platinated DNA was only moderately influenced by the protein redox state. Competition experiments involving p53CON in an oligonucleotide and non-specific oligonucleotides suggested that the p53 core domain is the primary site of (sc, lin and platinated) DNA binding in activated p53 while the p53 C-terminus is responsible for supercoil-selective DNA binding and recognition of platinated DNA in the latent p53. Considering the recently proposed role of the latent p53 in DNA repair (1) these observations may have implications in cell responses to DNA damage.

This work was supported by a grant of the Internal Grant Agency of the Ministry of Health of the Czech Rebublic No. NC/5343-3, and a grant of the Grant Agency of the Czech Republic No. 301/99/0692.

References and Footnotes

  1. Albrechtsen, N., Dornreiter, I., Grosse, F., Kim, E., Wiesmuller, L., and Deppert, W. (1999) Oncogene 18(53), 7706-17.
  2. Fojta, M., Brazdova, M., Cernocka, H., Pecinka, P., Brazda, V., Palecek, J., Jagelska, E., Vojtesek, B., Pospisilova, S., Subramaniam, V., Jovin, T. M., and Palecek, E. (2000) J. Biomol. Struct. Dyn. Conversation 11, 177-184
  3. .
  4. Fojta, M., Kubicarova, T., Vojtesek, B., and Palecek, E. (1999) J. Biol. Chem. 274(36), 25749-25755.
  5. Selivanova, G., Iotsova, V., Kiseleva, E., Strom, M., Bakalkin, G., Grafstrom, R. C., and Wiman, K. G. (1996) Nucleic Acids Research 24(18), 3560-7.

Miroslav Fojta, Marie Brazdova, Pavlina Becvarova, Jan Palecek, Sarka Pospisilova*, Borivoj Vojtesek* and Emil Palecek

Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno *Masaryk Memorial Cancer Institute, 656 53 Brno phone: +420-5-41517197, fax: +420-5-41211293, e-mail: fojta@ibp.cz