Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain.
The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings.
Mazur
S J
SJ
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA. mazurs@mail.nih.gov
Sakaguchi
K
K
Appella
E
E
Wang
X W
XW
Harris
C C
CC
Bohr
V A
VA
D000906:Antibodies / Q000494:pharmacology; D016367:Baculoviridae; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D000906:Antibodies; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004264:DNA Topoisomerases, Type I