Open access peer-reviewed chapter - ONLINE FIRST

A Study of p53 Action on DNA at the Single Molecule Level

Written By

Kiyoto Kamagata

Submitted: November 23rd, 2020 Reviewed: January 23rd, 2021 Published: March 1st, 2021

DOI: 10.5772/intechopen.96163

P53 - A Guardian of the Genome and Beyond Edited by Mumtaz Anwar

From the Edited Volume

P53 - A Guardian of the Genome and Beyond [Working Title]

Dr. Mumtaz Anwar, Dr. Zeenat Farooq, Associate Prof. Mohammad Tauseef and Dr. Vijay Avin Balaji Ragunathrao

Chapter metrics overview

227 Chapter Downloads

View Full Metrics


The transcription factor p53 searches for and binds to target sequences within long genomic DNA, to regulate downstream gene expression. p53 possesses multiple disordered and DNA-binding domains, which are frequently observed in DNA-binding proteins. Owing to these properties, p53 is used as a model protein for target search studies. It counters cell stress by utilizing a facilitated diffusion mechanism that combines 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs. Single-molecule fluorescence microscopy has been used to characterize individual motions of p53 in detail. In addition, a biophysical study has revealed that p53 forms liquid-like droplets involving the functional switch. In this chapter, the target search and regulation of p53 are discussed in terms of dynamic properties.


  • p53
  • single molecule
  • fluorescence
  • DNA
  • disordered
  • diffusion
  • jumping
  • intersegmental transfer
  • sliding
  • hopping
  • target search
  • liquid–liquid phase separation

1. Introduction

p53 is a multifunctional transcription factor that induces cell cycle arrest, DNA repair, and apoptosis, thereby suppressing cell cancerization [1, 2]. It is referred to as a guardian of the genome that determines cell fate. When p53 is activated by various stress factors, it searches for and binds to target DNA sequences and regulates the expression of downstream genes. p53 is composed of an N-terminal (NT) domain, core domain, linker, tetramerization (Tet) domain, and C-terminal (CT) domain. The core and Tet domains possess specifically folded structures, while other domains are intrinsically disordered [3, 4, 5]. p53 forms a tetramer via Tet domains [5]. Core and CT domains are involved in its binding to DNA sequences in a specific and nonspecific manner, respectively [6]. Fifty percent of gene mutations in tumor cells were found in p53, and many of the identified mutations were located in structured domains, which inhibited target DNA binding [3]. Comprehensive mutagenesis analysis supports the correlation between the structured domains of p53 and its function [7]. Since p53 possesses common properties frequently observed in DNA-binding proteins, including oligomerization, disordered regions, and multiple DNA-binding domains [8], it is used as a model protein in the target search study described below [9, 10, 11].

The target DNAs for p53 were ~ 20 bp, while the genomic DNA was ~109 bp. Accordingly, p53 was required to search for small targets efficiently from within vast lengths of non-target DNAs. This is known as a target search problem for sequence-specific DNA-binding proteins. To solve this problem, a facilitated diffusion mechanism has been proposed for DNA-binding proteins. The facilitated diffusion is the integration of three-dimensional (3D) diffusion in solution, one-dimensional (1D) diffusion along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs (Figure 1a). In 3D diffusion, p53 diffuses in solution, altering the search sites on genomic DNA. In 1D sliding, it moves along the DNA, while maintaining continuous contact. In addition, p53 hops or jumps along DNA (within 100 bp of jump). Intersegmental transfer enables p53 to move from one DNA to another without dissociation. Theoretical studies suggest that the integration of multiple search dynamics, while not requiring all dynamics, can facilitate the target search [14, 15, 16, 17]. The facilitation factor depends on various physical parameters, such as diffusion coefficient along DNA, residence time on DNA, dissociation time in solution, and frequency of transfer and jump.

Figure 1.

Target search dynamics of DNA-binding proteins and visualization of p53 dynamics on DNA by single-molecule fluorescence microscopy. (a) Schematic diagram of four target search dynamics. (b) Schematic diagram of single-molecule fluorescence microscope and flow cell. In the flow cell, one end of the DNA is tethered to the surface and it is stretched by buffer flow. p53 molecule labeled to a fluorescence dye is illuminated by TIRF and the fluorescence is detected by EM-CCD through an objective lens. Panels (a) and (b) are adapted from ref. [12] and ref. [13] with some modifications, respectively.

How does p53 solve the target search problem using facilitated diffusion? How is the target search and binding of p53 regulated? In this chapter, I explain the facilitated diffusion and regulation of p53 based on recently accumulated single-molecule data.


2. Single-molecule fluorescence microscopy

Single-molecule fluorescence microscopy enables the differentiation and characterization of individual search dynamics of DNA-binding proteins, including p53, as reported previously [18, 19, 20, 21, 22, 23, 24]. In general, the system combines a fluorescence microscope and a flow cell (Figure 1b). In the flow cell, one end of the DNA is tethered to the surface, and it is stretched by buffer flow. Several methods have been proposed for tethering DNAs [18, 25, 26, 27, 28, 29]. For example, a DNA garden is a simple method for producing DNA arrays, in which neutravidin molecules are printed in a line on polymer-coated coverslips, and biotinylated DNAs are tethered to the printed neutravidin [29]. p53 molecules labeled with a fluorescence dye are introduced into the flow cell using a syringe pump. The fluorescent p53 bound to DNA is selectively illuminated by total internal reflection fluorescence (TIRF). p53 molecules on DNA are detected as fluorescent spots on the sequential images of an electron-multiplying charge-coupled device (EM-CCD). The positions of molecules were tracked using an appropriate analysis program to visualize the search dynamics of p53.


3. Target search dynamics of p53

In 2008, 1D sliding of p53 along DNA was observed for the first time using single-molecule fluorescence microscopy [30]. This observation was consistent with a reported indirect evidence that p53 dissociated rapidly from short DNA in the absence of blocks at ends by sliding off from DNA [31]. In 2011, a study of p53 mutants deleting either of two DNA-binding proteins revealed that p53 can slide along DNA using disordered CT domains [32]. This is consistent with the fact that a designed peptide targeting CT domains suppressed the 1D sliding of p53 [33]. Furthermore, 1D sliding of p53 was supported by molecular dynamics simulations [34, 35]. In 2012, it was shown that 1D sliding dynamics of p53 depends slightly on DNA sequence, suggesting that p53 feels the energy landscape based on DNA sequence through interactions between core domains and DNA [36]. In 2015, a detailed analysis of 1D sliding dynamics demonstrated that p53 possesses two sliding modes on non-target DNA [37, 38]. In the fast mode, it interacts with DNA loosely using CT domains. In contrast, in the slow mode, it binds tightly to DNA using core and CT domains (Figure 2a). In 2017, the disordered linker was identified to trigger the switch between the two modes (Figure 2a) [41]. In 2016, the target recognition process of p53 was characterized in detail [42]. The results demonstrated that target recognition occurs mainly via 1D sliding. The target recognition of p53 was quite low (the successful recognition probability was 7%), but it was enhanced two-fold upon a post-translational modification. Accordingly, 1D sliding is considered as one of the important dynamics in the target search and binding of p53.

Figure 2.

Target search dynamics of p53. (a) Schematic diagram of two modes for 1D sliding p53 along DNA. p53 is composed of the NT (purple), Core (green), linker (black), Tet (yellow), and CT (pink) domains. The switch between two modes is triggered by the linker. (b) Typical single-molecule data showing intersegmental transfer of p53 between crisscrossing DNAs. (c) Schematic diagram of intersegmental transfer of p53 between two DNAs. p53 uses CT domains (pink) for the transfer. (d) Typical single-molecule data showing jumping of p53 along DNA (white traces). Arrows denote the jumping events. (e) Schematic diagram of encounter complex formation of p53 and conversion from the encounter complex to long-lived complex. Panel (b) is adapted from ref. [39] with some modifications. Panels (d) and (e) are adapted from ref. [40] with some modifications.

In 2018, intersegmental transfer of p53 was examined using ensemble kinetic and single-molecule fluorescence measurements [39]. After the solutions of p53 bound to fluorescently labeled DNA and non-labeled DNA were mixed, the transfer reaction of p53 was monitored between the two DNAs. The observed reactions included the dissociation of p53 from one DNA and its transfer to the other. Actually, as the concentration of non-labeled DNA increased, the observed rate constant increased, suggesting intersegmental transfer. The rate constant of the transfer was ~108 M−1 s−1, which is close to the diffusion limit. Furthermore, single-molecule tracking of p53 on crisscrossed DNAs demonstrated that p53 moves along the first DNA and then moves along the second DNA through the transfer at the intersection (Figure 2b). A study of p53 mutants deleting either of two DNA-binding domains identified that p53 binds to the first DNA and then to the second DNA using disordered CT domains at the same time; it then releases the first DNA, resulting in a transfer between the two DNAs (Figure 2c). This mechanism is supported by molecular dynamics simulations of p53 [43].

In 2020, the hopping/jumping of p53 on DNA was investigated [40]. Hopping/jumping was expected to occur at a time scale that is faster than the time resolution of the microscope (ex. 33 ms). To detect these events, the time resolution of the microscope was improved to 500 μs by optimizing the fluorescence excitation based on critical angle TIRF illumination and by utilizing the time delay integration mode of the EM-CCD [40]. Using the sub-millisecond-resolved microscope, jumping events of p53 along DNA were directly detected (arrows in Figure 2d). The jump frequency of p53 was ~6 s−1, and the jump time was 2.2 ms. Based on the study of p53 mutants deleting either of two DNA-binding domains, disordered CT domains were identified to be indispensable for the jumping of p53 along DNA [13]. Furthermore, 1D diffusion along DNA was enhanced upon increasing the salt concentration, suggesting that p53 moves along DNA by hopping DNA-binding domains. Thus, it was revealed that p53 possesses hopping and jumping dynamics along DNA.

In 2016, 3D diffusion of p53 was characterized using ensemble kinetic measurements [42]. Association rate constants for target and non-target DNAs were determined to be ~109 M−1 s−1, comparable to the diffusion limits. The difference in affinity for target and non-target DNAs was attributed to the dissociation rate constants. In 2020, the association process of p53 with non-target DNA was further investigated at the single-molecule level using a sub-millisecond resolved fluorescence microscope [40]. Kymographs demonstrated that short-lived traces of p53 with an average residence time of 2.8 ms were detected in addition to long-lived traces moving along DNA. The short-lived complex was interpreted as an encounter complex. Disordered CT domains of p53 were identified to participate in the transient complex formation and in the conversion from the transient complex to the long-lived complex [13] (Figure 2e). The long-lived complex was further stabilized by core domains [13].

Overall, single-molecule fluorescence microscopy revealed that p53 possesses all four search dynamics proposed theoretically: 3D diffusion, 1D sliding, hopping/jumping, and intersegmental transfer. The unique structure of p53, which is a tetramer of two DNA-binding domains, enables these search dynamics. This is the first study to examine all search possibilities for a single model protein.


4. Liquid–liquid phase separation of p53

Target search and binding of p53 might be regulated by a liquid-like assembly of p53 molecules. In a liquid–liquid phase separation (LLPS), p53 molecules, which disperse in the bulk phase, assemble and form a condensed phase called liquid droplets. In the droplet phase, p53 can move fluidly while maintaining a high concentration. This fluid property in the condensed phase differs from the solid aggregation that causes malfunction of p53. Early in vivostudies demonstrated that p53 is recruited into cellular droplets such as Cajal and promyelocytic leukemia protein (PML) bodies [44, 45, 46]. These facts suggest that LLPS might be involved in the cellular functions of p53.

In 2020, this possibility was extensively examined using in vitromeasurements such as scattering, DIC microscopy, and fluorescence microscopy [47]. p53 formed micrometer-sized droplets at neutral and slightly acidic pH and low salt concentrations. The fusion events of at least two droplets into a single large droplet were observed, confirming the fluidity of p53 inside the droplets (Figure 3a). High fluorescence intensity was detected in the droplets of p53 labeled with a fluorescent dye, supporting the high concentration of p53 in the droplets (Figure 3b). The droplet formation of p53 was affected by pH and salt concentrations. This suggests that attractive electrostatic interactions among local parts of p53 and repulsive net charges among whole molecules of p53 are balanced, resulting in droplet formation. Deletion of either of the disordered NT and CT domains suppressed the droplet formation of p53. This suggests multivalent electrostatic interactions between the oppositely charged NT and CT domains in p53 droplets.

Figure 3.

Liquid droplet formation of p53 regulates its function. (a) Time course of a typical fusion event of three p53 droplets into a single droplet using DIC microscopy. (b) DIC and fluorescence images of the droplets of Alexa488-labeled p53 and non-labeled p53. Scale bars in panels (a) and (b) represent 10 μm. (c) Schematic diagram of p53 conformation in the droplet. p53 is composed of the NT (purple), Core (orange), Tet (yellow), and CT (red) domains. In the droplets, the NT and CT domains interact electrostatically. Arrows denote the structural changes on the different domains of p53 that are induced by the intermolecular interactions in a droplet. The dimer structure is displayed for clarity. (d) Functional switch model of p53. The panels (a)-(d) are adapted from ref. [47] with some modifications.

The structural properties of p53 in solution and in droplet form were investigated using fluorescence resonance energy transfer (FRET) between two fluorophores labeled at two residues of p53. Since FRET depends on the distance between the two fluorophores, it was used to measure the conformational changes. The distance between the core domains of p53 was slightly longer in the droplets, while the distance between the CT domains became slightly shorter (Figure 3c). Accordingly, p53 adopted a new tertiary structure, forming interactions with the adjacent molecules in the droplets.

Does p53 maintain binding to the target DNA after experiencing the droplet formation? The reactions of p53 binding to the target DNA were similar before and after the droplet formation. These results indicate that droplet formation of p53 is reversible, and p53 dispersed in solution from the droplets retains its DNA binding ability.

Droplet formation of p53 was found to be regulated by molecular crowding, endogenous molecules, and post-translational modification. Molecular crowding agents, mimicking the cellular crowding condition, promoted droplet formation. In contrast, ssDNA, dsDNA, and ATP suppressed it. The p53 mutant mimicking post-translational phosphorylation did not form droplets. Based on these results, a functional switch model was proposed (Figure 3d). Under normal cell conditions, the compartmentalization of p53 into the droplets suppresses its function as a transcriptional regulator. Under stress conditions, the activation of p53, triggered by posttranslational phosphorylation, releases p53 from the droplets and promotes target search and binding.


5. Target search and regulation model of p53

In this section, the current model of p53 is described in terms of target search and regulation. p53 functions as a transcription factor that responds to various emergency situations in cells. Under normal cell conditions, p53 turns off through the following mechanisms. First, the copy number of p53 is maintained at a low level, allowing dimers with a low affinity to target DNAs in an oligomeric state [48, 49]. Second, post-translational modifications for activating p53 are not added, for example, suppressing the target recognition of p53 [42]. Third, p53 is stored in liquid droplets [47]. These actions of p53 prevent its malfunction under normal conditions.

Under cellular stress, p53 is activated by post-translational modifications [1, 2, 50, 51, 52, 53, 54, 55, 56] and by a change in its oligomeric state from dimers to tetramers, with a high affinity for target DNAs [48, 57, 58, 59]. Phosphorylation of the CT domain of p53 triggers its release from the droplets, allowing it to engage in target search [47]. The increase in the copy number of p53 also facilitates the target search [60]. As explained above, p53 utilizes facilitated diffusion combining four search dynamics. Using 3D diffusion, p53 associates randomly with nonspecific sites of DNA, followed by dissociation. Until p53 associates with the target sequence by chance, it repeats such association and dissociation motions. If the search motion of p53 is limited to 3D diffusion, it would be a time-consuming endeavor. After p53 associates with the nonspecific site of DNA by 3D diffusion, it can search for the target sequence along DNA near the bound site using 1D sliding and hopping/jumping. The search distance of p53 per association event is estimated to be 700 bp [40], corresponding to approximately 35-fold facilitation of the target search.

In cells, genomic DNAs are covered by many DNA-binding proteins, including histones and other nucleoid proteins. These DNA-binding proteins may act as obstacles in the target search of p53. For example, when the sliding p53 collides with other DNA-binding proteins on DNA, it may not be able to bypass these obstacles due to steric hindrance, thereby limiting the search distance on DNA. However, p53 possesses two bypass mechanisms: the jumping along DNA [40] and the intersegmental transfer between two DNAs [39]. Using these motions, it can overcome such obstacles and continue its search for targets in cells. Overall, the search and regulation strategies of p53 could satisfy various cellular requirements.


6. Conclusions

The target search and binding of p53 and its regulation have been characterized using single-molecule fluorescence microscopy and relevant biophysical methods. The accumulated data demonstrate that p53 searches for target DNAs utilizing four search dynamics: 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs (Figure 4). Especially, hopping/jumping and intersegmental transfer between two DNAs are required to bypass obstacles bound to DNA. It was reported that other DNA-binding protein with a disordered DNA-binding domain bypasses obstacles through obstacle-unbound region of DNA [24]. Since p53 possesses a similar disordered DNA-binding domain, it is not surprising that p53 possesses this bypass mechanism. Target search and binding are regulated by copy number, post-translational modifications, and liquid droplet formation. Considering that p53 can interact with many partner proteins, the partner proteins may affect the target search. Complexity in the target search and regulation of p53 would enable a response to various emergency situations in cells and be required to satisfy various cellular requirements.

Figure 4.

Schematic diagram of target search of p53. Pink and gray circles are p53 and obstacle bound to DNA, respectively. Four search mechanisms are illustrated.



I thank the corroborators for their helpful discussions on our studies of target search by p53.


  1. 1. Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-mediated tumour suppression. Nature Reviews Cancer. 2014:14:359-370. DOI: 10.1038/nrc3711
  2. 2. Beckerman R, Prives C. Transcriptional regulation by p53. Cold Spring Harbor Perspectives in Biology. 2010:2:a000935. DOI: 10.1101/cshperspect.a000935
  3. 3. Joerger AC, Fersht AR. The tumor suppressor p53: from structures to drug discovery. Cold Spring Harbor Perspectives in Biology. 2010:2:a000919. DOI: 10.1101/cshperspect.a000919
  4. 4. Laptenko O, Tong DR, Manfredi J, Prives C. The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein. Trends in Biochemical Sciences. 2016:41:1022-1034. DOI: 10.1016/j.tibs.2016.08.011
  5. 5. Kamada R, Toguchi Y, Nomura T, Imagawa T, Sakaguchi K. Tetramer formation of tumor suppressor protein p53: Structure, function, and applications. Biopolymers. 2016:106:598-612. DOI: 10.1002/bip.22772
  6. 6. Anderson ME, Woelker B, Reed M, Wang P, Tegtmeyer P. Reciprocal interference between the sequence-specific core and nonspecific C-terminal DNA binding domains of p53: Implications for regulation. Molecular and Cellular Biology. 1997:17:6255-6264. DOI: 10.1128/MCB.17.11.6255
  7. 7. Kato S, Han SY, Liu W, Otsuka K, Shibata H, Kanamaru R, Ishioka C. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proceedings of the National Academy of Sciences of the United States of America. 2003:100:8424-8429. DOI: 10.1073/pnas.1431692100
  8. 8. Vuzman D, Levy Y. Intrinsically disordered regions as affinity tuners in protein-DNA interactions. Molecular Biosystems. 2012:8:47-57. DOI: 10.1039/c1mb05273j
  9. 9. Tafvizi A, Mirny LA, van Oijen AM. Dancing on DNA: kinetic aspects of search processes on DNA. Chemphyschem. 2011:12:1481-1489. DOI: 10.1002/cphc.201100112
  10. 10. Kamagata K, Murata A, Itoh Y, Takahashi S. Characterization of facilitated diffusion of tumor suppressor p53 along DNA using single-molecule fluorescence imaging. J Photochem Photobiol C Photochem Reviews. 2017:30:36-50. DOI: 10.1016/j.jphotochemrev.2017.01.004
  11. 11. Kamagata K, Itoh Y, Subekti DRG. How p53 molecules solve the target DNA search problem: a review. International Journal of Molecular Sciences. 2020:21. DOI: 10.3390/ijms21031031
  12. 12. Kamagata K. Front line of DNA-binding protein research (Japanese). Precision Medicine. 2020:3:542-548. DOI:
  13. 13. Subekti DRG, Kamagata K. The disordered DNA-binding domain of p53 is indispensable for forming an encounter complex to and jumping along DNA. Biochemical and Biophysical Research Communications. 2021:534:21-26. DOI: 10.1016/j.bbrc.2020.12.006
  14. 14. Sheinman M, Kafri Y. The effects of intersegmental transfers on target location by proteins. Physical Biology. 2009:6:016003. DOI: 10.1088/1478-3975/6/1/016003
  15. 15. Bauer M, Metzler R. Generalized facilitated diffusion model for DNA-binding proteins with search and recognition states. Biophysical Journal. 2012:102:2321-2330. DOI: 10.1016/j.bpj.2012.04.008
  16. 16. Veksler A, Kolomeisky AB. Speed-Selectivity Paradox in the Protein Search for Targets on DNA: Is It Real or Not?, Journal of Physical Chemistry B. 2013:117:12695-12701. DOI: 10.1021/jp311466f
  17. 17. Mahmutovic A, Berg OG, Elf J. What matters for lac repressor search in vivo--sliding, hopping, intersegment transfer, crowding on DNA or recognition?, Nucleic Acids Research. 2015:43:3454-3464. DOI: 10.1093/nar/gkv207
  18. 18. Kabata H, Kurosawa O, Arai I, Washizu M, Margarson SA, Glass RE, Shimamoto N. Visualization of single molecules of RNA-polymerase sliding along DNA. Science. 1993:262:1561-1563. DOI:
  19. 19. Blainey PC, Luo G, Kou SC, Mangel WF, Verdine GL, Bagchi B, Xie XS. Nonspecifically bound proteins spin while diffusing along DNA. Nature Structural & Molecular Biology. 2009:16:1224-1229. DOI: 10.1038/nsmb.1716
  20. 20. Visnapuu ML, Greene EC. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nature Structural & Molecular Biology. 2009:16:1056-1062. DOI: 10.1038/nsmb.1655
  21. 21. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2014:507:62-67. DOI: 10.1038/nature13011
  22. 22. Nelson SR, Dunn AR, Kathe SD, Warshaw DM, Wallace SS. Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases. Proceedings of the National Academy of Sciences of the United States of America. 2014:111:E2091–E2099. DOI: 10.1073/pnas.1400386111
  23. 23. Kamagata K, Mano E, Ouchi K, Kanbayashi S, Johnson RC. High free-energy barrier of 1D diffusion along DNA by architectural DNA-binding proteins. Journal of Molecular Biology. 2018:430:655-667. DOI: 10.1016/j.jmb.2018.01.001
  24. 24. Kamagata K, Ouchi K, Tan C, Mano E, Mandali S, Wu Y, Takada S, Takahashi S, Johnson RC. The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA. Nucleic Acids Research. 2020:48:10820-10831. DOI: 10.1093/nar/gkaa799
  25. 25. Harada Y, Funatsu T, Murakami K, Nonoyama Y, Ishihama A, Yanagida T. Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophysical Journal. 1999:76:709-715. DOI: 10.1016/S0006-3495(99)77237-1
  26. 26. Fazio T, Visnapuu ML, Wind S, Greene EC. DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging. Langmuir. 2008:24:10524-10531. DOI: 10.1021/la801762h
  27. 27. Visnapuu ML, Fazio T, Wind S, Greene EC. Parallel arrays of geometric nanowells for assembling curtains of DNA with controlled lateral dispersion. Langmuir. 2008:24:11293-11299. DOI: 10.1021/la8017634
  28. 28. Hughes CD, Wang H, Ghodke H, Simons M, Towheed A, Peng Y, Van Houten B, Kad NM. Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic Acids Research. 2013:41:4901-4912. DOI: 10.1093/nar/gkt177
  29. 29. Igarashi C, Murata A, Itoh Y, Subekti DRG, Takahashi S, Kamagata K. DNA garden: a simple method for producing arrays of stretchable DNA for single-molecule fluorescence imaging of DNA-binding proteins. Bulletin of the Chemical Society of Japan. 2017:90:34-43. DOI: 10.1246/bcsj.20160298
  30. 30. Tafvizi A, Huang F, Leith JS, Fersht AR, Mirny LA, van Oijen AM. Tumor suppressor p53 slides on DNA with low friction and high stability. Biophysical Journal. 2008:95:L01–L03. DOI: 10.1529/biophysj.108.134122
  31. 31. McKinney K, Mattia M, Gottifredi V, Prives C. p53 linear diffusion along DNA requires its C terminus. Molecular Cell. 2004:16:413-424. DOI: 10.1016/j.molcel.2004.09.032
  32. 32. Tafvizi A, Huang F, Fersht AR, Mirny LA, van Oijen AM. A single-molecule characterization of p53 search on DNA. Proceedings of the National Academy of Sciences of the United States of America. 2011:108:563-568. DOI: 10.1073/pnas.1016020107
  33. 33. Kamagata K, Mano E, Itoh Y, Wakamoto T, Kitahara R, Kanbayashi S, Takahashi H, Murata A, Kameda T. Rational design using sequence information only produces a peptide that binds to the intrinsically disordered region of p53. Scientific Reports. 2019:9:8584. DOI: 10.1038/s41598-019-44688-0
  34. 34. Khazanov N, Levy Y. Sliding of p53 along DNA can be modulated by its oligomeric state and by cross-talks between its constituent domains. Journal of Molecular Biology. 2011:408:335-355. DOI: 10.1016/j.jmb.2011.01.059
  35. 35. Terakawa T, Kenzaki H, Takada S. p53 searches on DNA by rotation-uncoupled sliding at C-terminal tails and restricted hopping of core domains. Journal of the American Chemical Society. 2012:134:14555-14562. DOI: 10.1021/ja305369u
  36. 36. Leith JS, Tafvizi A, Huang F, Uspal WE, Doyle PS, Fersht AR, Mirny LA, van Oijen AM. Sequence-dependent sliding kinetics of p53. Proceedings of the National Academy of Sciences of the United States of America. 2012:109:16552-16557. DOI: 10.1073/pnas.1120452109
  37. 37. Murata A, Ito Y, Kashima R, Kanbayashi S, Nanatani K, Igarashi C, Okumura M, Inaba K, Tokino T, Takahashi S, Kamagata K. One-dimensional sliding of p53 along DNA is accelerated in the presence of Ca(2+) or Mg(2+) at millimolar concentrations. Journal of Molecular Biology. 2015:427:2663-2678. DOI: 10.1016/j.jmb.2015.06.016
  38. 38. Murata A, Itoh Y, Mano E, Kanbayashi S, Igarashi C, Takahashi H, Takahashi S, Kamagata K. One-dimensional search dynamics of tumor suppressor p53 regulated by a disordered C-terminal domain. Biophysical Journal. 2017:112:2301-2314. DOI: 10.1016/j.bpj.2017.04.038
  39. 39. Itoh Y, Murata A, Takahashi S, Kamagata K. Intrinsically disordered domain of tumor suppressor p53 facilitates target search by ultrafast transfer between different DNA strands. Nucleic Acids Research. 2018:46:7261-7269. DOI: 10.1093/nar/gky586
  40. 40. Subekti DRG, Murata A, Itoh Y, Takahashi S, Kamagata K. Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking. Scientific Reports. 2020:10:13697. DOI: 10.1038/s41598-020-70763-y
  41. 41. Subekti DRG, Murata A, Itoh Y, Fukuchi S, Takahashi H, Kanbayashi S, Takahashi S, Kamagata K. The disordered linker in p53 participates in nonspecific binding to and one-dimensional sliding along DNA revealed by single-molecule fluorescence measurements. Biochemistry. 2017:56:4134-4144. DOI: 10.1021/acs.biochem.7b00292
  42. 42. Itoh Y, Murata A, Sakamoto S, Nanatani K, Wada T, Takahashi S, Kamagata K. Activation of p53 facilitates the target search in DNA by enhancing the target recognition probability. Journal of Molecular Biology. 2016:428:2916-2930. DOI: 10.1016/j.jmb.2016.06.001
  43. 43. Takada S, Kanada R, Tan C, Terakawa T, Li W, Kenzaki H. Modeling Structural Dynamics of Biomolecular Complexes by Coarse-Grained Molecular Simulations. Accounts of Chemical Research. 2015:48:3026-3035. DOI: 10.1021/acs.accounts.5b00338
  44. 44. Fogal V, Gostissa M, Sandy P, Zacchi P, Sternsdorf T, Jensen K, Pandolfi PP, Will H, Schneider C, Del Sal G. Regulation of p53 activity in nuclear bodies by a specific PML isoform. EMBO Journal. 2000:19:6185-6195. DOI: 10.1093/emboj/19.22.6185
  45. 45. Guo A, Salomoni P, Luo J, Shih A, Zhong S, Gu W, Pandolfi PP. The function of PML in p53-dependent apoptosis. Nature Cell Biology. 2000:2:730-736. DOI: 10.1038/35036365
  46. 46. Cioce M, Lamond AI. Cajal bodies: a long history of discovery. Annual Review of Cell and Developmental Biology. 2005:21:105-131. DOI: 10.1146/annurev.cellbio.20.010403.103738
  47. 47. Kamagata K, Kanbayashi S, Honda M, Itoh Y, Takahashi H, Kameda T, Nagatsugi F, Takahashi S. Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains. Scientific Reports. 2020:10:580. DOI: 10.1038/s41598-020-57521-w
  48. 48. Gaglia G, Guan Y, Shah JV, Lahav G. Activation and control of p53 tetramerization in individual living cells. Proceedings of the National Academy of Sciences of the United States of America. 2013:110:15497-15501. DOI: 10.1073/pnas.1311126110
  49. 49. Weinberg RL, Veprintsev DB, Fersht AR. Cooperative binding of tetrameric p53 to DNA. Journal of Molecular Biology. 2004:341:1145-1159. DOI: 10.1016/j.jmb.2004.06.071
  50. 50. Hamard PJ, Lukin DJ, Manfredi JJ. p53 basic C terminus regulates p53 functions through DNA binding modulation of subset of target genes. Journal of Biological Chemistry. 2012:287:22397-22407. DOI: 10.1074/jbc.M111.331298
  51. 51. Hamard PJ, Barthelery N, Hogstad B, Mungamuri SK, Tonnessen CA, Carvajal LA, Senturk E, Gillespie V, Aaronson SA, Merad M, Manfredi JJ. The C terminus of p53 regulates gene expression by multiple mechanisms in a target- and tissue-specific manner in vivo. Genes and Development. 2013:27:1868-1885. DOI: 10.1101/gad.224386.113
  52. 52. Marouco D, Garabadgiu AV, Melino G, Barlev NA. Lysine-specific modifications of p53: a matter of life and death?, Oncotarget. 2013:4:1556-71. DOI:
  53. 53. Laptenko O, Shiff I, Freed-Pastor W, Zupnick A, Mattia M, Freulich E, Shamir I, Kadouri N, Kahan T, Manfredi J, Simon I, Prives C. The p53 C terminus controls site-specific DNA binding and promotes structural changes within the central DNA binding domain. Molecular Cell. 2015:57:1034-1046. DOI: 10.1016/j.molcel.2015.02.015
  54. 54. Retzlaff M, Rohrberg J, Kupper NJ, Lagleder S, Bepperling A, Manzenrieder F, Peschek J, Kessler H, Buchner J. The regulatory domain stabilizes the p53 tetramer by intersubunit contacts with the DNA binding domain. Journal of Molecular Biology. 2013:425:144-155. DOI: 10.1016/j.jmb.2012.10.015
  55. 55. Friedler A, Veprintsev DB, Freund SM, von Glos KI, Fersht AR. Modulation of binding of DNA to the C-terminal domain of p53 by acetylation. Structure. 2005:13:629-636. DOI: 10.1016/j.str.2005.01.020
  56. 56. Loffreda A, Jacchetti E, Antunes S, Rainone P, Daniele T, Morisaki T, Bianchi ME, Tacchetti C, Mazza D. Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity. Nat Commun. 2017:8:313. DOI: 10.1038/s41467-017-00398-7
  57. 57. Kawaguchi T, Kato S, Otsuka K, Watanabe G, Kumabe T, Tominaga T, Yoshimoto T, Ishioka C. The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library. Oncogene. 2005:24:6976-6981. DOI: 10.1038/sj.onc.1208839
  58. 58. Rajagopalan S, Huang F, Fersht AR. Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53. Nucleic Acids Research. 2011:39:2294-2303. DOI: 10.1093/nar/gkq800
  59. 59. Fischer NW, Prodeus A, Malkin D, Gariepy J. p53 oligomerization status modulates cell fate decisions between growth, arrest and apoptosis. Cell Cycle. 2016:15:3210-3219. DOI: 10.1080/15384101.2016.1241917
  60. 60. Wang YV, Wade M, Wong E, Li YC, Rodewald LW, Wahl GM. Quantitative analyses reveal the importance of regulated Hdmx degradation for P53 activation. Proceedings of the National Academy of Sciences of the United States of America. 2007:104:12365-12370. DOI: 10.1073/pnas.0701497104

Written By

Kiyoto Kamagata

Submitted: November 23rd, 2020 Reviewed: January 23rd, 2021 Published: March 1st, 2021