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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

p53在模拟核环境的DNA上的靶序列搜索机制：通过单分子观察阐明

基本信息

批准号：
19J11761
负责人：
SUBEKTI Dwiky
金额：
$ 1.09万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2019
资助国家：
日本
起止时间：
2019-04-25 至 2021-03-31
项目状态：
已结题

项目摘要

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).

在2020-2021财年，我继续使用亚毫秒单分子荧光显微镜（SMFM）对p53沿着DNA扩散动力学进行成像的研究。如前一年所报道的，我在25 mM KCl至150 mM KCl的各种盐浓度下进行了成像。在不同的盐浓度下，我发现p53始终表现出两种不同的结合模式，对应于其结合寿命的两种不同分布。观察到的>88%的p53分子表现出非常短的结合，寿命为2-3 ms，我将这一群体称为瞬时结合。剩下的12%更稳定地结合DNA，并表现出跳跃和一维扩散。讨论瞬时结合、跳跃和1D扩散的结果被总结并发表在Sci.此外，我还使用具有截短的C-末端结构域（CoreTet-p53）和截短的核心结构域（TetCT-p53）的不同p53突变体进行了实验。在125 mM KCl浓度下，停留时间分布呈双指数衰减。时间常数分别为2.053 ± 0.009 ms（98% ± 1%）和15.1 ± 0.4 ms（2% ± 1%）。作为参考，全长p53（FL-p53）在相同条件下的停留时间分布表现出类似的双指数衰减，时间常数为1.70 ± 0.12（96%± 3%）和20.7 ± 2.7ms（4% ± 2%）。结果表明，CT结构域在瞬时结合的形成中起重要作用，而核心结构域稳定了长寿命复合物。该结果总结在BBRC第534卷，21-26中。（2021年）。