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Elucidation of the correlation between oligomer formation, DNA unwinding, and chemo-mechanical coupling of a helicase

Research Project

Project/Area Number 18K06169
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 43040:Biophysics-related
Research InstitutionThe Graduate School for the Creation of New Photonics Industries

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)

Project Period (FY) 2018-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
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)
Keywords1分子計測 / ナノバイオ / 核酸 / 酵素反応 / 1分子計測
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.

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。

Report

(5 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Research-status Report
  • 2019 Research-status Report
  • 2018 Research-status Report
  • Research Products

    (20 results)

All 2022 2021 2020 2019 2018 Other

All Journal Article (7 results) (of which Peer Reviewed: 7 results,  Open Access: 6 results) Presentation (10 results) (of which Int'l Joint Research: 1 results,  Invited: 5 results) Remarks (3 results)

  • [Journal Article] Quantitative and kinetic single-molecule analysis of DNA unwinding by <i>Escherichia coli</i> UvrD helicase2022

    • Author(s)
      Hiroaki Yokota
    • Journal Title

      Biophysics and Physicobiology

      Volume: 19 Issue: 0 Pages: n/a

    • DOI

      10.2142/biophysico.bppb-v19.0006

    • ISSN
      2189-4779
    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Development of MEMS Mirror-Type Laser Microdissection2021

    • Author(s)
      長谷川 正仁、工 藤 靖、平野 美奈子、横田 浩章
    • Journal Title

      Transactions of Japanese Society for Medical and Biological Engineering

      Volume: 59 Issue: 4-5 Pages: 95-103

    • DOI

      10.11239/jsmbe.59.95

    • NAID

      130008124747

    • ISSN
      1347-443X, 1881-4379
    • Year and Date
      2021-09-10
    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] DNA Unwinding and Oligomerization Dynamics of <i>Escherichia coli</i> UvrD Helicase Revealed by Single-molecule Fluorescence Imaging2021

    • Author(s)
      横田 浩章
    • Journal Title

      Seibutsu Butsuri

      Volume: 61 Issue: 4 Pages: 227-231

    • DOI

      10.2142/biophys.61.227

    • NAID

      130008068992

    • ISSN
      0582-4052, 1347-4219
    • Related Report
      2021 Annual Research Report 2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD2021

    • Author(s)
      Yokota Hiroaki
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 22 Issue: 3 Pages: 1018-1018

    • DOI

      10.3390/ijms22031018

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Low-Light Photodetectors for Fluorescence Microscopy2021

    • Author(s)
      Yokota Hiroaki、Fukasawa Atsuhito、Hirano Minako、Ide Toru
    • Journal Title

      Applied Sciences

      Volume: 11 Issue: 6 Pages: 2773-2773

    • DOI

      10.3390/app11062773

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] DNA-unwinding dynamics of Escherichia coli UvrD lacking the C-terminal 40 amino acids2020

    • Author(s)
      Yokota, H.
    • Journal Title

      Biophys. J.

      Volume: 118 Issue: 7 Pages: 1634-1648

    • DOI

      10.1016/j.bpj.2020.02.014

    • Related Report
      2020 Research-status Report 2019 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Fluorescence microscopy for visualizing single-molecule protein dynamics2019

    • Author(s)
      Yokota Hiroaki
    • Journal Title

      Biochimica et Biophysica Acta (BBA) - General Subjects

      Volume: in press Issue: 2 Pages: 129362-129362

    • DOI

      10.1016/j.bbagen.2019.05.005

    • Related Report
      2019 Research-status Report 2018 Research-status Report
    • Peer Reviewed
  • [Presentation] MEMSミラー型レーザーマイクロダイセクション装置の開発2021

    • Author(s)
      長谷川 正仁、工 藤 靖、平野 美奈子、横田 浩章
    • Organizer
      第17回バイオオプティクス研究会
    • Related Report
      2021 Annual Research Report
    • Invited
  • [Presentation] DNA-unwinding dynamics of Escherichia coli UvrD lacking C-terminal amino acids2021

    • Author(s)
      Hiroaki Yokota
    • Organizer
      第59回日本生物物理学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] DNA-unwinding dynamics of Escherichia coli UvrD lacking the C-terminal 40 amino acids2021

    • Author(s)
      Hiroaki Yokota
    • Organizer
      Biophysical Society 65th Annual Meeting
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Presentation] DNA-unwinding dynamics of Escherichia col UvrD lacking the C-terminal 40 amino acids2020

    • Author(s)
      Hiroaki Yokota
    • Organizer
      第58回日本生物物理学会年会
    • Related Report
      2020 Research-status Report
  • [Presentation] DNA-unwinding dynamics of Escherichia coli UvrD lacking the C-terminal 40 amino acids2020

    • Author(s)
      Hiroaki Yokota
    • Organizer
      第43回日本分子生物学会年会
    • Related Report
      2020 Research-status Report
  • [Presentation] 生体分子1 分子の蛍光イメージング~DNA結合タンパク質の1 分子直視~2019

    • Author(s)
      横田 浩章
    • Organizer
      第16回バイオオプティクス研究会
    • Related Report
      2019 Research-status Report
    • Invited
  • [Presentation] Single-molecule imaging of a non-hexameric Escherichia coli helicase UvrD mutant lacking C-terminal residues2019

    • Author(s)
      横田 浩章
    • Organizer
      第57回日本生物物理学会年会
    • Related Report
      2019 Research-status Report
    • Invited
  • [Presentation] 生体分子1分子の蛍光イメージングと蛍光検出~1分子直視と高時間分解能検出~2018

    • Author(s)
      横田 浩章
    • Organizer
      第15回バイオオプティクス研究会
    • Related Report
      2018 Research-status Report
    • Invited
  • [Presentation] 大腸菌非六量体型DNAヘリカーゼUvrD変異体の1分子イメージング2018

    • Author(s)
      横田 浩章
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Research-status Report
    • Invited
  • [Presentation] Single-molecule imaging of the oligomeric form of the non-hexameric Escherichia coli helicase UvrD mutants2018

    • Author(s)
      横田 浩章
    • Organizer
      第56回日本生物物理学会年会
    • Related Report
      2018 Research-status Report
  • [Remarks]

    • URL

      https://www.gpi.ac.jp/research/bpxd/professor-15/

    • Related Report
      2021 Annual Research Report
  • [Remarks] 光産業創成大学院大学バイオフォトニクスデザイン分野ホームページ

    • URL

      https://www.gpi.ac.jp/research/bpxd/professor-15/

    • Related Report
      2020 Research-status Report
  • [Remarks] 光産業創成大学院大学光バイオ分野ホームページ

    • URL

      https://www.gpi.ac.jp/research/bio/professor-15/

    • Related Report
      2019 Research-status Report

URL: 

Published: 2018-04-23   Modified: 2023-01-30  

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