Supercoil-Selective Binding of Protein p53: Influence of the Protein Redox State and Roles of p53 Domains
Full length wild type tumor suppressor protein p53 (fl p53) preferentially binds to supercoiled (sc) DNA regardless of the presence or absence of the p53 consensus sequence (1,2). This supercoil-selective (SCS) binding was partially inhibited by the protein oxidation by diamide; under the same conditions, p53 sequence-specific DNA binding was completely abolished (3). Cysteine residues (moieties oxidizable by diamide) are located exclusively in the p53 core (the domain responsible for sequence-specific DNA binding) (4). Another, non-specific DNA binding site is situated at the protein C-terminus. DNA binding through the C-terminal domain was not inhibited by the protein oxidation (5). Considering these facts, the roles of the two protein domains in the SCS binding of reduced (p53red) and oxidized (p53ox) p53 were studied using truncated p53 molecules and monoclonal antibodies (MAb).
Complex of pBluescript SK scDNA with fl p53red was supershifted by the MAb DO-1 (binding to the p53 N-terminal domain) as well as by MAbs Bp53-10 and Bp53-30 (recognizing an epitope at the protein C-terminus) (6,7). On the other hand, scDNA complex with p53ox was supershifted by DO-1 while Bp53-30 (6) and Bp53-10 inhibited SCS binding p53ox-scDNA complex formation. Protein p53 lacking last 30 amino acid residues at the C-terminus (CD30 p53) significantly bound scDNA in its reduced but not oxidized state. Similar behavior was observed also with isolated p53 core domain (CD p53). In competition experiments involving sc and linear DNA of pBluescript SK(-), fl p53red exhibited strong binding for the scDNA. On the contrary, CD30 p53, CD p53 (8) and fl p53 bound by the MAb Bp53-10 exhibited only negligible preference for the scDNA. Under the same conditions, fl p53ox (without the MAb) bound scDNA displaying about 10 to 20-fold preference (by an order of magnitude lower, as compared to that of fl p53red).
Based on the above data, it can be concluded that the primary site responsible for the strong SCS p53 DNA binding is the protein C-terminus. Those p53 constructs lacking free C-terminus (the truncated ones, or the fl protein with C-terminus blocked by the MAbs) in the reduced state bind sc and linear DNAs with similar affinities. The essential DNA binding site of these proteins is most probably the core domain. Upon oxidation, DNA-binding activity of the p53 core domain is abolished. On the other hand, fl p53ox (possessing intact C-terminus) still can bind scDNA with a decreased but significant preference. The p53 C-terminal DNA-binding site probably cooperate with the protein core domain in the strong SCS DNA binding of fl p53red.
This work was supported by grants of the Internal Grant Agency of the Ministry of Health of the Czech Republic No. NC 5343-3 and of the Grant Agency of the Czech Republic No. 301/99/0692.
1. E. Palecek, D. Vlk, V. Stankova, V. Brazda, B. Vojtesek, T. R. Hupp, A. Schaper and T. M. Jovin, Oncogene 15, 2201-2209 (1997).
Miroslav Fojta, Marie Brazdova, Pavlina Becvarova, Jan Palecek, Sarka Pospisilova2, Borivoj Vojtesek2, and Emil Palecek
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno 2Masaryk Memorial Cancer Institute, Brno