Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA

Abstract

p53 is an anti-tumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using Atomic Force Microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultra-low noise solidstate nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labelling is either not possible or at least not a viable option.

Imaging and kinetics of the bimolecular complex formed by the tumor suppressor p53 with ubiquitin ligase COP1 as studied by atomic force microscopy and surface plasmon resonance

“…While bimolecular spots (the right arrow), whose representative cross-section profile is shown in the right inset ( Figure 3B), likely correspond to dimers of p53, in agreement with literature data that reported the presence of p53 dimers at similar concentrations. 33 The height of the p53 images is significantly lower than that obtained for COP1 molecules, according to the lower MW of p53 protein with respect to COP1.…”

Investigating the Influence of Magnesium Ions on p53–DNA Binding Using Atomic Force Microscopy

“…The tumor suppressor protein p53 is a widely distributed phosphoprotein that is the central player in the pathways controlling cell growth, DNA repair, cell differentiation, senescence, and apoptosis [ 1 , 2 , 3 , 4 , 5 , 6 ]. It is found that over 50% of human cancers are associated mutations in the p53 protein [ 1 ].…”

“…The tumor suppressor protein p53 is a widely distributed phosphoprotein that is the central player in the pathways controlling cell growth, DNA repair, cell differentiation, senescence, and apoptosis [ 1 , 2 , 3 , 4 , 5 , 6 ]. It is found that over 50% of human cancers are associated mutations in the p53 protein [ 1 ]. The p53 protein is a flexible multidomain protein containing 393 residues in full length, and consists of four distinct domains: (a) the unstructured N-terminal trans-activation domain (N-ter) and a proline-rich domain, which can bind to a series of proteins and regulates p53 transcription [ 7 ] and dissociation from DNA [ 8 ]; (b) the central core region known as the DNA-binding domain (DBD), which regulates the specific binding to DNA [ 9 ]; (c) the tetramerization domain (Tet) and (d) the C-terminal domain (C-Ter) which has been shown to bind nonspecifically to DNA [ 1 , 10 ].…”

High-speed detection of DNA translocation in nanopipettes

“…It does not rule out that the observed scaling behavior is a property of the polymer itself, but the present model does offer an explanation that reconciles differences in scaling factors observed with the same analyte (DNA), but with different platforms or experimental contexts. In fact, surface effects have been shown to have a significant impact on the translocation of proteins and protein/DNA complexes, 36,37 and it is also well-known that the interaction between DNA and a surface is strongly dependent on the electrolyte composition. 38 To this end, the presence of Mg 2+ results in the adsorption of DNA to mica, while preserving some strand mobility when bound to the surface (i.e.…”