p53 Specifically Binds Triplex DNA In Vitro and in Cells.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Triplex DNA structures are involved in gene regulation and genomic instability.
• The tumor suppressor p53 protein recognizes DNA sequences and structures to control cell fate.
• Understanding p53's interaction with non-canonical DNA structures is crucial for deciphering its regulatory roles.

Purpose of the Study:

• To investigate the structure-specific binding of the p53 protein to T.A.T. DNA triplexes.
• To determine the effect of p53 binding to triplex DNA on p53-driven transcription.
• To identify p53 target genes regulated through T.A.T. triplex DNA interactions.

Main Methods:

• In vitro DNA binding assays with full-length p53 and deletion variants.
• Competition assays, immunoprecipitation, and atomic force microscopy (AFM) for triplex recognition.
• Chromatin immunoprecipitation (ChIP) for in vivo binding analysis.
• Luciferase reporter assays and RT-qPCR for transcriptional activity assessment.

Main Results:

• p53 binds to both intermolecular and intramolecular T.A.T. triplex DNA structures.
• The C-terminal DNA binding domain of p53 is essential for triplex recognition.
• p53 binding to T.A.T. triplexes was confirmed in vitro and in vivo.
• Insertion of triplex-forming sequences enhanced p53-mediated reporter gene transactivation.
• A set of candidate p53 target genes with both consensus and T.A.T. motifs were identified, with several showing p53-dependent activation.

Conclusions:

• T.A.T. DNA triplexes represent a novel class of binding sites for the p53 protein.
• p53 recognizes and binds to T.A.T. triplexes in a DNA structure-dependent manner.
• This structure-specific DNA binding by p53 contributes to the regulation of gene transcription in cells.