2019 Fiscal Year Annual Research Report
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 Institution | Tohoku University |
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Principal Investigator |
SUBEKTI Dwiky 東北大学, 理学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2019-04-25 – 2021-03-31
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| Keywords | p53 / Single Molecule / protein-DNA interaction / Fluorescence imaging / Sub-millisecond Imaging |
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| Outline of Annual Research Achievements |
I have conducted the research on imaging of p53 diffusion dynamics along DNA using a sub-millisecond single-molecule fluorescence microscope (SMFM). 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.
Analyzing the data taken across different salt concentration, the average jump frequency (f) and averaged diffusion coefficient (D) both showed salt dependency. From the data taken at 150 mM KCL, f value of jump is 6 jump/second and D value of 1D diffusion is 1.4 micrometer^2/second. Both f and D values at 150 mM are roughly 4 times higher than those obtained at 25 mM KCl. The plot of f against D at various salt concentrations showed a strong correlation of 0.85. Considering the salt-dependent 1D diffusion along DNA and correlation between f and D, I propose that p53 searches for the target DNA with non-rotational coupled motion where it combines 1D sliding with small jumps (hopping). I hypothesize that the interaction between individual DNA binding domain of p53 with DNA is important in determining the movement of p53 on DNA such as hopping and jumping. The result of this research is currently under submission in academic journal.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In this study, we aimed to measure the dynamics of p53 along DNA under in-vivo mimicking condition using sub-millisecond single molecule fluorescence microscope that we developed. In the beginning of FY 2019, however, we faced challenges in obtaining good signal to noise ratio images and the tracking of such particles. We managed to resolve the issues by optimizing the experiment protocol and the particle track analysis program we used. As a result, we managed to identify transient binding and other dynamics. We tried numerous experiments to confirm our observation and currently trying to publish our result. Although our main purpose has not been fulfilled yet, we consider our discoveries as important step for the future direction of this study. Therefore, we think that the research is progressing steadily towards our purpose.
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| Strategy for Future Research Activity |
In the FY2020, we will continue to conduct single-molecule fluorescence imaging at sub-millisecond time resolution. Our main priority at the moment would be publishing our recent results in the journal as well as progressing to the in vivo mimic experiments.
Currently, we are preparing lambda-DNA and nucleosome assembly arrays that will be used in the in vivo mimic experiments. We are also preparing the microscope system so that we can conduct two color experiments. With two different dye, we will be able to visualize p53 and the nucleosome assembly simultaneously. With such system, we expect that we will be able to obtain many important dynamics of p53 as it moves around DNA and nucleosomes.
Using the two color system, we may also prepare p53 dimers with different fluorescent dyes and observe how the FRET occur between the dye pair. Such experiment will give us important insights on p53's molecular structure during its target search dynamics.
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