Binding of Full Length p53 and its Core Domain to Supercoiled DNA
We showed (1) that the full-length human wild type of p53 (fl p53) bound preferentially to negatively supercoiled DNA at native superhelix density regardless of presence or absence of the consensus sequence and we described conditions under which this binding is inhibited or stimulated (1-5). We investigated the core domain (p53CD, aa 94-312) of human p53 binding to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Insect expressed fl (p53i) and bacterially-expressed p53 (p53b) showed only small differences in binding to scDNA. Comparison of the binding of fl p53 and p53CD indicated however significant differences (6); p53CD produced a relatively small retardation of scDNA in contrast to the ladder of bands formed by fl p53 (both by p53i and p53b) in agarose gels. Competition between scDNAs and their linearized (lin) forms showed preferences for scDNAs but the ratios characterizing distribution of the protein between sc and lin DNAs were in pBluescript DNA substantially higher for fl p53 (sc/lin ~ 60) than for p53CD (sc/lin ~ 4). Due to the strong sequence specific binding of p53 to lin pPGM1 DNA (containing the consensus sequence, p53CON) the preference of this protein for sc pPGM1 DNA was lower but still significant. Strong binding of fl p53 to scDNA not containing p53CON represents a new p53 binding mode which we tentatively call supercoil-specific (SCS) binding. This binding requires in addition to the core domain other region(s) of the p53 molecule, most likely a region of the C-terminal domain. DNA segments defined both by the nucleotide sequence and their spatial arrangement and/or strand crossings (7) or bending in scDNAs may be involved in p53 binding. The binding preference of p53CD for scDNA may be due to non-specific binding to internal single-stranded regions in scDNA (absent in relaxed DNA molecules) and/or to a weakened SCS binding.
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).
Marie Brazdova1, Sabina Billova1, Vaclav Brazda1, Jan Palecek1, Emil Palecek1, Vinod Subramaniam1 and Thomas M. Jovin1
Institute of Biophysics, Academy of Sciences of the Czech Republic,