One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg(2+) and Ca(2+) after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg(2+) or Ca(2+), indicating that the divalent cations accelerate the sliding likely by weakening the DNA-p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg(2+) and Ca(2+), which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.

early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function. Tumor suppressor p53 is a multifunctional transcription factor that induces cell cycle arrest, DNA repair or apoptosis upon binding to its target DNA sequence. In 50% of human cancers, mutations on p53 are found to hamper its binding to the target sequence. Accordingly, extensive investigations have been conducted to characterize the functions as well as malfunctions of p53. However, an important aspect of p53, namely its involvement in liquid-like droplets, is still largely unresolved. In fact, p53 has long been known to be uptaken into cellular droplets such as Cajal and promyelocytic leukemia protein (PML) bodies. In this report, we describe that p53 itself can form liquid-like droplets upon the control of solution conditions, suggesting a possible involvement of the p53 droplets in the cellular environment. The primary function of p53 is the accommodation of various posttranslational modifications, termed activation, which in turn triggers the search for and the binding to its target DNA sequence, leading to the expression of downstream genes 1. p53 is composed of the N-terminal (NT) (residues 1-95), the core (95-293), the linker (293-326), the tetramerization (Tet) (326-357), and the C-terminal (CT) (357-393) domains. p53 slides along nonspecific DNA by attaching the CT domain to the DNA and by hopping the core domain 2-4. The sliding of p53 occurs in two modes 5,6 , in which the CT, core and linker domains are differently in contact with the DNA 7. The recognition efficiency of the target sequence by the sliding p53 is low, but is enhanced by the activation of p53 8. Furthermore, the sliding p53 can transfer from one DNA strand to another using the CT domain 9. In addition,

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.

Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca and Mg, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg]. Although binding of CoreTet to DNA becomes markedly weaker at higher [Mg], binding of TetCT depends slightly on [Mg]. Single-molecule fluorescence measurements revealed that the one-dimensional diffusion of CoreTet along DNA consists of fast and slow search modes, the ratio of which depends strongly on [Mg]. In contrast, diffusion of TetCT consisted of only the fast mode. The disordered C-terminal domain can associate with DNA irrespective of [Mg], and can maintain an equilibrium balance of the two search modes and the p53 search distance. These results suggest that p53 modulates the quaternary structure of the complex between p53 and DNA under different [Mg] and that it maintains the target search along DNA.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host–guest interactions hinder the understanding of the molecular grammar governing liquid–liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.