| 研究開始時の研究の概要 |
Using a newly developed Single Molecule Fluorescence Microscopy with sub-millisecond time-resolution, this study investigates the target search mechanism and obstacle avoidance mechanism of tumor suppressor protein p53 in nuclear mimicking condition. The nuclear mimicking condition is achieved by preparing DNA-histone complex array designed to mimic chromatin (tightly compacted DNA), partially unwrapped DNA, and fully unwrapped DNA.
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| 研究実績の概要 |
In FY 2020-2021, I continued my research on imaging of p53 diffusion dynamics along DNA using a sub-millisecond single-molecule fluorescence microscope (SMFM). As reported in the previous year, I conducted the imaging under various salt concentration ranging from 25 mM KCl up to 150 mM KCl. Under the varying salt concentration, I found that p53 consistently exhibits two different binding modes corresponding to two different distribution of its binding lifetime. >88% of p53 molecules observed exhibited a very short binding with the lifetime of 2-3 ms, I refer to this population as transient binding. The remaining 12% binds DNA more stably and exhibit jumps and 1D diffusion. The result discussing the transient binding, jump, and 1D diffusion were summarized and published in Sci. Rep. 10, 13697 (2020).
In addition, I also conducted experiments using different p53 mutants with truncated C-terminal domain (CoreTet-p53) and truncated core domain (TetCT-p53). At 125 mM KCl concentration, The residence time distribution showed a double exponential decay. The time constants were 2.053 ± 0.009 ms (98% ± 1%) and 15.1 ± 0.4 ms (2% ± 1%), respectively. As a reference, the residence time distribution of full-length p53 (FL-p53) under the same condition demonstrated a similar double exponential decay with time constants of 1.70 ± 0.12 (96%± 3%) and 20.7 ± 2.7ms (4% ± 2%). The result suggests that CT domain are important in the formation of transient binding and that the core domain stabilizes the long-lived complex. This result was summarized in BBRC Vol. 534, 21-26. (2021).
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