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 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 absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress.
Fojta
Miroslav
M
Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-612 65 Brno, Czech Republic. fojta@ibp.cz
Pivonkova
Hana
H
Brazdova
Marie
M
Nemcova
Katerina
K
Palecek
Jan
J
Vojtesek
Borivoj
B
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004278:DNA, Superhelical / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D004278:DNA, Superhelical; D016159:Tumor Suppressor Protein p53