TAILIEUCHUNG - Báo cáo khoa học: Investigations of the supercoil-selective DNA binding of wild type p53 suggest a novel mechanism for controlling p53 function

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodiesmapping toepitopes at theproteinC-terminus (to block binding within the CTDBS) | Eur. J. Biochem. 271 3865-3876 2004 FEBS 2004 doi Investigations of the supercoil-selective DNA binding of wild type p53 suggest a novel mechanism for controlling p53 function Miroslav Fojta1 Hana Pivonkova1 Marie Brazdova1 2 Katerina Nemcova1 Jan Palecek1 3 and Borivoj Vojtesek4 1 Laboratory of Biophysical Chemistry and Molecular Oncology Institute of Biophysics Academy of Sciences of the Czech Republic Brno Czech Republic department of Tumor Virology Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg Hamburg Germany 3Genome Damage and Stability Centre University of Sussex Falmer Brighton UK 4Masaryk Memorial Cancer Institute Brno Czech Republic The tumor suppressor protein p53 selectively binds to supercoiled sc DNA lacking the specific p53 consensus binding sequence p53CON . Using p53 deletion mutants we have previously shown that the p53 C-terminal DNA-binding site CTDBS is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues to preclude binding within the p53 core domain and or by antibodies mapping to epitopes at the protein C-terminus to block binding within the CTDBS . In the absence of antibody reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective SCS binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the

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