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.
- MeSH
- lidé MeSH
- monoklonální protilátky metabolismus MeSH
- mutageneze cílená MeSH
- nádorový supresorový protein p53 metabolismus ultrastruktura MeSH
- oxidace-redukce MeSH
- sekvenční delece MeSH
- superhelikální DNA metabolismus ultrastruktura MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa protilátek MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- MeSH
- aktiny chemie metabolismus MeSH
- cytoskeletální proteiny genetika chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- glykoproteiny chemie MeSH
- lidé MeSH
- myši MeSH
- proteiny intermediálních filament genetika chemie metabolismus MeSH
- vazebná místa MeSH
- vimentin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- fosfatidylinositoly metabolismus MeSH
- fosfolipidy biosyntéza genetika metabolismus MeSH
- genetická transkripce MeSH
- kyseliny fosfatidové metabolismus MeSH
- proteiny přenášející fosfát fyziologie genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u hub MeSH
- techniky in vitro MeSH
