核内環境を模したＤＮＡ上におけるｐ５３の標的配列探索機構：一分子観察による解明
| Project/Area Number |
19J11761
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Single-year Grants |
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| Section | 国内 |
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| Review Section |
Basic Section 43040:Biophysics-related
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| Research Institution | Tohoku University |
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Principal Investigator |
SUBEKTI Dwiky 東北大学, 多元物物質科学研究所, 特別研究員(PD)
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| Project Period (FY) |
2019-04-25 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2020: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2019: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Sub-millisecond imaging / p53 / encounter complex / Single Molecule / protein-DNA interaction / Fluorescence imaging / Sub-millisecond Imaging |
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| Outline of Research at the Start |
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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| Outline of Annual Research Achievements |
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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| Research Progress Status |
令和2年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和2年度が最終年度であるため、記入しない。
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Report
(2 results)
Research Products
(7 results)
