Tumor Suppressor p53 Binds with High Affinity to CTG{middle dot}CAG Trinucleotide Repeats and Induces Topological Alterations in Mismatched Duplexes

Walter K, Warnecke G, Bowater R, Deppert W and Kim EL
Source: J Biol Chem
Publication Date: (2005)
Issue: 280(52): 42497-42507
Research Area:
Cancer Research/Cell Biology
Nucleofector® I/II/2b
Used a subclone of LNZ308 (LNZ308/2024) which expresses wild type p53 in the presence of doxycycline. Transfected a plasmid expressing luciferase under the control of the mdm2(P2) promoter (intact, with p53-binding sites deleted or with p53-binding sites deleted and with CTG-CAG repeats inserted) in presence or absence of doxycycline in order to assess tanscriptional activity of p53 binding CTG-CAG repeats.
DNA binding is central to the ability of p53 to function as a tumor suppressor. In line with the remarkable functional versatility of p53, which can act on DNA as a transcription, repair, recombination, replication, and chromatin accessibility factor, the modes of p53 interaction with DNA are also versatile. One feature common to all modes of p53-DNA interaction is the extraordinary sensitivity of p53 to the topology of its target DNA. Whereas the strong impact of DNA topology has been demonstrated for p53 binding to sequence-specific sites or to DNA lesions, the possibility that DNA structure-dependent recognition may underlie p53 interaction with other types of DNA has not been addressed until now. We demonstrate for the first time that conformationally flexible CTG.CAG trinucleotide repeats comprise a novel class of p53-binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in vivo. Our major finding is that p53 binds to CTG.CAG tracts by different modes depending on the conformation of DNA. Although p53 binds preferentially to hairpins formed by either CTG or CAG strands, it can also bind to linear forms of CTG.CAG tracts such as canonic B DNA or mismatched duplex. Intriguingly, by binding to a mismatched duplex p53 can induce further topological alterations in DNA, indicating that p53 may act as a DNA topology-modulating factor.