Sequence-Specific Binding of Tumour Suppressor Protein p53 to DNA. Effect of DNA Supercoiling
Mutations in the TP53 gene are one of the most frequent genetic alterations observed in human cancer (in about 50% of human malignancies). The human 53kDa nuclear phosphoprotein p53 is a transcription factor capable of inhibiting the growth of tumour cells transformed by oncogenes, presumably by virtue of its checkpoint activity(1). The p53 protein binds to promoters and activates trancription of related genes p21and gadd45 arresting cell cycle in G1 or G2 phase. During this time the cell can start the process of reparation of DNA damage. If damage of DNA is too extensive and enzymes cannot control reparation process, the p53 protein can induce apoptosis. The p53 protein consists of three functional domains: N-terminal domain which contains proline-rich region and a subdomain responsible for transcription-activating function. Core domain is the site of sequence-specific DNA binding of p53 protein and renders suppression of oncogene transformation. Most of point mutations related to cancer occur in this part of p53 protein. The C-terminal domain assembles stable tetramers and is responsible for the non-specific binding to single and double-strand DNA?s.
We showed that p53 protein was preferentially bound to negatively supercoiled DNA (scDNA) (2). We found that some metal ions and oxidation agents inhibited this binding (2-5). Negative supercoiling of scDNA can influence p53 DNA binding at least in two ways: (a) by enhancement of p53 binding to the sites not containing p53CON whose spatial arrangement is changed in favour of p53 binding, or (b) by affecting the binding to p53CON. Recently it has been shown (6) that the sequence specific DNA binding of p53 to the mdm2 promoter is inhibited in vitro when this promoter is present in scDNA. On the other hand sequence-specific DNA binding to p21 promoter was not inhibited by DNA supercoiling. We cloned p53CON sequences derived from promoters mdm2, p21 and RGC as well as a synthetic p53CON (5?-AGACATGCCTAGACATGCCT-3?) (2) into pBluescript DNA and developed a competition assay for p53 binding to p53CON in scDNAs. We found that DNA supercoiling at native superhelix density stimulates p53 to the tested p53CONs. Insertion of (TA)8T (forming a cruciform in scDNA) into the mdm2 sequence resulted in a decrease of p53 binding but not in its full elimination.
1.Deppert, W.,Seminars in Cancer Biol. 5, 187-203 (1994).
E. Jagelska , L. Karlovska and E. Palecek
Institute of Biophysics, Academy of Science of the Czech Republic,