Development of a novel multiplex in vitro binding  assay to profile p53-DNA interactions.
The p53 tumor suppressor plays a critical role in cancer biology, functioning as a transcription factor capable of directing cell fate. It interacts with specific DNA response elements (REs) to regulate the activity of target genes. We describe here a novel, non-radioactive assay to measure p53-DNA binding which involves the sequential use of in vitro transcription/ translation (IVT), immunoprecipitation and real-time PCR. The method reliably enables the detection of sequence-specific DNA binding of full-length p53 at low concentrations of physiologically relevant REs (<5 nM). Furthermore, we demonstrate multiplexing of 4 different REs in a single binding reaction. The use of IVT precludes the requirement for purified protein, enabling rapid characterization of the binding properties of p53 variants. Uniquely, it also offers the opportunity to add compounds during translation that might modulate and activate p53. When compared to prevailing protein-DNA binding assays, this method exhibits comparable or higher sensitivity, in addition to an expansive dynamic range afforded by the use of real-time PCR. A further extrapolation of its utility is demonstrated when the addition of a peptide known to activate p53 increased its binding to a consensus RE, consistent with published data.
Goh
Walter
W
p53 Laboratory, Immunos, Singapore.
Lane
David
D
Ghadessy
Farid
F
D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D050505:Mutant Proteins; D010455:Peptides; D016159:Tumor Suppressor Protein p53; D004247:DNA