Elucidation of the correlation between oligomer formation, DNA unwinding, and chemo-mechanical coupling of a helicase
| Project/Area Number |
18K06169
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 43040:Biophysics-related
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| Research Institution | The Graduate School for the Creation of New Photonics Industries |
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Principal Investigator |
Yokota Hiroaki 光産業創成大学院大学, 光産業創成研究科, 准教授 (90415547)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | 1分子計測 / ナノバイオ / 核酸 / 酵素反応 / １分子計測 |
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| Outline of Final Research Achievements |
Single-molecule visualization was performed for the non-hexameric superfamily 1 helicase UvrD protein, which plays a crucial role in DNA repair in E. coli.
Single-molecule direct visualization of the C-terminal 40 amino acid deletion mutant UvrDΔ40C, which was used in studies that proposed the monomeric model for DNA unwinding, revealed that two or three UvrDΔ40C molecules were simultaneously involved in DNA unwinding, possibly in an oligomeric form, similar to that with wild-type UvrD. On the other hand, single-molecule direct visualization of a UvrD mutant that exhibits enhanced DNA unwinding activity revealed that the number of the mutant bound to DNA was higher than that of wild-type UvrD.
In addition, to visualize DNA unwinding by UvrD with single-nucleotide resolution, a DNA substrate for the visualization was prepared, and the visualization was tested. Moreover, using zero-mode waveguides, attempts were made to visualize association/dissociation of ATP with/from UvrD.
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| Academic Significance and Societal Importance of the Research Achievements |
酵素や受容体など多くのタンパク質で見られる多量体形成は、タンパク質の活性制御、構造安定性の増大など、その機能や構造に重要な役割を果たす。一方、タンパク質複合体のダイナミクスに迫るのが困難なこともあり、タンパク質間の相互作用や多量体形成と機能の関係については不明な点が多く残されている。
本研究では、多量体形成がDNA巻き戻し機能に重要な役割を果たしている大腸菌のDNA修復タンパク質ヘリカーゼUvrDに注目した。そして、生体1分子間の相互作用を観察できる蛍光1分子イメージングにより、そのDNA上での多量体形成・DNA巻き戻し機能・ATP加水分解エネルギーの化学力学共役機構の相関関係に迫った。
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Report
(5 results)
Research Products
(20 results)
