@article{03be02a5a3544fa8b4d32522dfeddf34,
title = "Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking",
abstract = "Characterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the long-lived p53–DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging.",
author = "Subekti, {Dwiky Rendra Graha} and Agato Murata and Yuji Itoh and Satoshi Takahashi and Kiyoto Kamagata",
note = "Funding Information: The time resolution may be improved using higher excitation power to increase the rate of photon emission and limiting the area of the imaging detector to increase the frame rate. The rate of photon detection from single fluorophores can be easily increased by increasing the excitation laser power at the expense of the total observation period owing to photobleaching. The imaging area can be reduced significantly by considering the one dimensionality of the stretched DNA and by restricting the region of interest by a slit. In the standard mode of EM-CCD, fluorescence photons from single molecules are accumulated as electrons in each pixel of the two-dimensional detector, which are sent and read out by an A/D converter one by one. However, by limiting the observation area to one stretched DNA, the time delay integration (TDI) detection mode of the EM-CCD, provided by Hamamatsu Photonics, can enhance the frame rate up to ~ 100 fold and achieve sub-millisecond detection of the dynamics of DNA-binding proteins. p53 is a transcription factor that suppresses the cancerization of cells and has been investigated as a representative DNA-binding protein demonstrating facilitated diffusion mainly based on single-molecule fluorescence measurements8,17–24. p53 binds to DNA nonspecifically and slides along DNA to search the target DNA sequence17–19. p53 possesses two sliding modes having different contacts with DNA by two DNA binding domains: core and C-terminal (CT) domains20,21. The disordered linker enables switching between the two sliding modes22. p53 slides along DNA rotationally following the DNA groove15,25. Target recognition by the sliding p53 is quite low and can be regulated by single mutations8. In addition to the sliding, p53 can utilize the ultrafast intersegmental transfer between two DNAs, which could help to skip obstacles bound to DNA during the target search in cells23. These dynamics of p53 were partly supported by molecular dynamics simulations26–29. Thus, single-molecule fluorescence studies have improved our understanding of the facilitated diffusion of p53. Publisher Copyright: {\textcopyright} 2020, The Author(s).",
year = "2020",
month = dec,
day = "1",
doi = "10.1038/s41598-020-70763-y",
language = "English",
volume = "10",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",
}