Study of regulation of DNA replication by controlling superhelicities of single DNA molecules

• Project/Area Number: 25650002
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Molecular biology
• Research Institution: Gunma University
• Principal Investigator: Shinji Katsura 群馬大学, 大学院理工学府, 教授 (10260598)
• Co-Investigator(Kenkyū-buntansha): Oshige Masahiko 群馬大学, 大学院理工学府, 准教授 (00451716)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: １分子観察 / DNA２次構造 / 超らせん / マグネチックツィーザー / 磁気ピンセット / 微細流路 / １本鎖DNA / DNA複製制御 / ＤＮＡ２次構造 / ＤＮＡ複製 / ＤＮＡ形態制御 / ＤＮＡ超らせん

Outline of Final Research Achievements

We developed an apparatus based on magnetic tweezers to introduce supercoil on a single DNA molecule, and studied the effects of negative superhelicities on the local DNA denaturation in λDNA containing the SV40 origin of DNA replication. Furthermore, we also studied the effects of superhelicities on the local denaturation in the presence of SV40 Large T antigen（SV40TAｇ）, which is an initiation protein of SV40 DNA replication,
Even in the absence of SV40TAｇ, the local denaturation of SV40ori-λDNA under a negative supercoil state was observed around the replication origin and the probability of occurrence of the local denaturation increased with negative superhelicity. Furthermore, addition of SV40TAｇ enhanced the probability of occurrence of the local denaturation and extended the single-stranded region under higher negative superhelicities.

Research Products

• [Journal Article] Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State. (2015)
  • Author(s): Takahashi S, Motooka S, Usui T, Kawasaki S, Miyata H, Kurita H, Mizuno T, Matsuura S, Mizuno A, Oshige M, Katsura S.
  • Journal Title: Analytical Chemistry Volume: 87 Issue: 6 Pages: 3490-3497
  • DOI: 10.1021/acs.analchem.5b00044
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel (2014)
  • Author(s): S. Takahashi, T. Usui, S. Kawasaki, H. Miyata, H. Kurita, S.-i. Matsuura, A. Mizuno, M. Oshige, and S. Katsura
  • Journal Title: Analytical Biochemistry Volume: - Pages: 24-30
  • DOI: 10.1016/j.ab.2014.04.012
  • Peer Reviewed
• [Journal Article] A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A (2014)
  • Author(s): S. Takahashi, S. Kawasaki, H. Miyata, H. Kurita, T. Mizuno, S.-i. Matsuura, A. Mizuno, M. Oshige, and S. Katsura
  • Journal Title: Sensors Volume: 14 Issue: 3 Pages: 5174-5182
  • DOI: 10.3390/s140305174
  • Peer Reviewed
• [Journal Article] Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel (2013)
  • Author(s): S. Takahashi, S. Kawasaki, K. Yamaguchi, H. Miyata, H. Kurita, T. Mizuno, S. Matsuura, A. Mizuno, M. Oshige, and S. Katsura
  • Journal Title: Journal of Fluorescence Volume: - Issue: 4 Pages: 635-640
  • DOI: 10.1007/s10895-013-1210-1
  • Peer Reviewed
• [Presentation] 蛍光複製タンパク質を用いた１本鎖DNA標識によるDNA合成反応の１分子観察 (2015)
  • Author(s): 高橋 俊介、石川裕一、柳基成、小和瀬聡実、川崎祥平、栗田弘史、水野武、松浦俊一、水野彰、大重真彦、桂進司
  • Organizer: BMB2015(第38回日本分子生物学会年会･第88回日本生化学会大会 合同大会)
  • Place of Presentation: 神戸ポートアイランド(兵庫県神戸市)
  • Year and Date: 2015-12-01
• [Presentation] DNAおよびDNA代謝酵素の1分子蛍光観察による動態解析 (2015)
  • Author(s): 高橋俊介、川崎祥平、栗田弘史、水野武、松浦俊一、水野彰、大重真彦、桂進司
  • Organizer: 化学工学会群馬大会2015
  • Place of Presentation: 桐生市民文化会館（群馬県桐生市）
  • Year and Date: 2015-11-27
• [Presentation] Direct Single-Molecule Observations of DNA Synthesis and Digestion Reactions (2015)
  • Author(s): Takahashi, S., Kawasaki, S., Kurita, H., Mizuno, T., Matsuura, S-I., Mizuno, A., Oshige, M., Katsura, S.
  • Organizer: The 11th International Workshop on Radiation Effects on Semiconductor Devices for Space Applications & The 7th International Conference on Advanced Micro-Device Engineering
  • Place of Presentation: City Performing Arts Center (Kiryu, Gunma, Japan)
  • Year and Date: 2015-11-11
  • Int'l Joint Research
• [Presentation] 負の超らせん状態でのSV40ラージT抗原によるDNA解鎖の１分子観察 (2015)
  • Author(s): 高橋俊介、川崎祥平、栗田弘史、水野武、松浦俊一、花岡文雄、水野彰、大重真彦、桂進司
  • Organizer: 第２３回DNA複製・組換え・修復ワークショップ
  • Place of Presentation: 焼津グランドホテル(静岡県焼津市)
  • Year and Date: 2015-10-19
• [Presentation] 負の超らせんDNAによる二重らせんの局所的な開裂の1分子観察 (2015)
  • Author(s): 高橋俊介、本岡伸也、川崎祥平、栗田弘史、水野武、松浦俊一、水野彰、大重真彦、桂進司
  • Organizer: 2015年度生物工学若手研究者の集い（若手会）夏のセミナー
  • Place of Presentation: タナベ名古屋研修センター（愛知県北名古屋市）
  • Year and Date: 2015-07-11
• [Presentation] Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel, (2014)
  • Author(s): S. Takahashi, T. Usui, S. Kawasaki, H. Miyata, H. Kurita, S. Matsuura, A. Mizuno, M. Oshige, S. Katsura
  • Organizer: 1st International Symposium of Gunma University Medical Innovation and 6th International Conference on Advanced Micro-Device Engineering (GUMI&AMDE2014)
  • Place of Presentation: Kiryu, Gunma, JAPAN
  • Year and Date: 2014-12-05
• [Presentation] 負の超らせん導入による二重らせんの局所的な開裂の1分子直接観測 (2014)
  • Author(s): 高橋俊介, 本岡伸也, 川崎祥平, 宮田英史, 栗田弘史, 松浦俊一, 水野武, 水野彰, 大重真彦, 桂進司
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜、神奈川
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel (2013)
  • Author(s): S. Takahashi, S. Kawasaki, K. Yamaguchi, H. Miyata, H. Kurita, T. Mizuno, S. Matsuura, A. Mizuno, M. Oshige and S, Katsura
  • Organizer: 5th International Conference on Advanced Micro-Device Engineering
  • Place of Presentation: Kiryu, Gunma, Japan
• [Presentation] 蛍光複製タンパク質A を用いた１本鎖DNA 標識によるDNA 合成反応のリアルタイム１分子観察 (2013)
  • Author(s): 高橋 俊介，川崎 祥平，宮田 英史，栗田 弘史，水野 武，松浦 俊一，水野 彰，大重 真彦，桂 進司
  • Organizer: 第３６回日本分子生物学会
  • Place of Presentation: 神戸ポートアイランド