Abstract
The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble.
Highlights
The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site
Crystal structures of the isolated DNA binding domain (DBD) bound to a single DNA half site revealed two DBD monomers bound per half site, with two such dimer/DNA complexes packing into tetramers, leading to the model that p53 binds DNA as a dimer of dimers[28]
To understand mechanisms by which DNA binding occurs we developed a single molecule fluorescence assay to quantify full length tetrameric p53 binding to DNA in vitro
Summary
The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. P53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. P53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble. The contributions of regions such as the N-terminal and C-terminal domains of p53 to parameters such as binding affinity, kinetic stability, and oligomeric state, are unclear This is important in light of more recent evidence showing that regions of p53 outside of the DNA binding domain can impact the interaction of p53 with D NA15–20. Lower resolution EM studies with full length p53 suggest a p53 tetramer can bind a single half site, and that an octamer can assemble on the DNA if p53 tetramers bind both half sites present in an RE30–32
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