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

Yang Chen,Guangcan Yang,Yanwei Wang,Tianyong Gao

doi:10.3390/ijms18071585

Abstract

p53 is a tumor suppressor protein that plays a significant role in apoptosis and senescence, preserving genomic stability, and preventing oncogene expression. Metal ions, such as magnesium and zinc ions, have important influences on p53–DNA interactions for stabilizing the structure of the protein and enhancing its affinity to DNA. In the present study, we systematically investigated the interaction of full length human protein p53 with DNA in metal ion solution by atomic force microscopy (AFM). The p53–DNA complexes at various p53 concentrations were scanned by AFM and their images are used to measure the dissociation constant of p53–DNA binding by a statistical method. We found that the dissociation constant of p53 binding DNA is 328.02 nmol/L in physiological buffer conditions. The influence of magnesium ions on p53–DNA binding was studied by AFM at various ion strengths through visualization. We found that magnesium ions significantly stimulate the binding of the protein to DNA in a sequence-independent manner, different from that stimulated by zinc. Furthermore, the high concentrations of magnesium ions can promote p53 aggregation and even lead to the formation of self-assembly networks of DNA and p53 proteins. We propose an aggregation and self-assembly model based on the present observation and discuss its biological meaning.

Highlights

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]
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]. p53 DBD is a sequence-specific transcription factor which is activated in response to a variety of DNA damaging agents, and p53 signaling may suppress apoptosis and induce senescence [5,6]
We found that divalent magnesium ions significantly stimulated the binding of the protein to DNA in a sequence-independent manner, and high concentrations of magnesium ions can promote p53 aggregation and lead to the self-assembly networks of DNA and p53 proteins

Summary

Introduction

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]. The C-Ter and Tet are of critical importance for this mode They contribute to the fast, nonspecific association of p53 and DNA; (b) The 1D sliding mode. Jumping is repeating dissociation and re-association from one DNA site to another or from one port to another port on a single DNA stretch [2,21,22]; (d) The intersegmental transfer mode. In this mode, the protein relocates from one site of DNA to another site by heterotrimeric complex formation [14]. The p53–DNA binding can be a model system to shed light on protein binding mechanisms since it is a key protein subject to facilitated diffusion, where both the 3D diffusion and 1D sliding of p53 have been demonstrated by single-molecule fluorescence microscopy [14,19]

Methods

Results

Conclusion