Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

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.

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

“…To do so we asked if binding and dissociation events involved the gain or loss of two dye molecules en route to the final bound or unbound state. As shown in the bar plots in Figure 2B, on each of the four REs, the vast majority of the events showed tetrameric binding and dissociation that did not proceed through an intermediate (gray bars), consistent with what we observed in our previous experiments with the GADD45 RE 12 . Moreover, there is no significant difference between the REs regarding the percentage of each type of transition (i.e.…”

“…oligomeric state) of p53 bound to DNA. Figure is adapted from 12 . ( B ) Representative AF647 emission data from a single DNA molecule with five tetrameric p53 binding events.…”

“…Emission movies of DNA+p53 were analyzed as described using in-house software written in IDL 12 . Briefly, spots in the DNA-only movie and the DNA+p53 movie from the same region of a slide were computationally colocalized.…”

“…Indeed, we observed that the p53/RE interactions were surprisingly dynamic, with many binding and release events occurring over tens of seconds. Moreover, p53 tetramers dissociated from the native RE with two different rate constants, showing that two kinetically distinguishable populations of p53/DNA complexes exist 12 . Notably, single particle tracking experiments in live cells have also shown that p53 interacts with chromatin with rapid dynamics.…”

Adaptive homeostasis and the p53 isoform network

“…As well as the affinity and specificity of p53 proteins for the different p53RE sequences, they are also influenced by their number, as most p53‐inducible genes contain clusters of p53REs separated by 0–13 bp. This enables oligomerization and stacking of p53 proteins on DNA (Kern et al , 1991 ; Stenger et al , 1994 ; Bourdon et al , 1997 ; Vyas et al , 2017 ; Lei et al , 2019 ; Ly et al , 2020 ; Senitzki et al , 2021 ). Thus, the large variety of p53 REs and permutation of p53 isoforms allow the p53 network to regulate the expression of a broad range of genes to maintain and restore cell and organ function and thus organismal integrity (Beno et al , 2011 ; Afek et al , 2020 ; Farkas et al , 2021 ).…”