Development of novel method of Zero Mode Waveguides for characterization of DNA binding proteins

• Project/Area Number: 25440008
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Kyoto University
• Principal Investigator: HAN YONG-WOON 京都大学, 物質－細胞統合システム拠点, 助教 (50566297)
• Research Collaborator: IWASA Takuma 京都大学, 大学院生命科学研究科, 大学院生
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 蛍光１分子イメージング / DNA結合タンパク質

Outline of Final Research Achievements

Zero Mode Waveguides (ZMWs) consist of nanosized holes in an aluminum film. Thus, ZMWs enable us the visualization of a single fluorescent molecule under micromolar order of the molecules. We characterized E. coli RuvB motor protein, which promotes Holliday junction DNA branch migration, and SRA, hemi-methyl CpG binding domain. We observed fluorescently labeled RuvBs binding to a RuvA-Holliday junction under various nucleotide conditions and our data suggested that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation

Research Products

• [Journal Article] The application of fluorescence-conjugated pyrrole/imidazole polyamides in the characterization of protein-DNA complex formation (2016)
  • Author(s): Han YW, Sugiyama H, Harada Y
  • Journal Title: Biomater Sci. Volume: 4 Issue: 3 Pages: 391-399
  • DOI: 10.1039/c5bm00214a
  • Peer Reviewed / Open Access
• [Journal Article] ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues. (2015)
  • Author(s): Oomoto, I., Suzuki-Hirano, A., Umeshima, H., Han, YW., Yanagisawa, H., Carlton, P., Harada, Y., Kengaku, M., Okamoto, A., Shimogori, T. and Wang, DO.
  • Journal Title: Nucleic Acids Res. Volume: 43(19) Issue: 19 Pages: e126-e126
  • DOI: 10.1093/nar/gkv614
  • NAID: 120005619785
  • Peer Reviewed / Open Access
• [Journal Article] Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation. (2015)
  • Author(s): Iwasa, T., Han, Y. W., Hiramatsu, R., Yokota, H., Nakao, K., Yokokawa, R., Ono, T., Harada, Y.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 18177-18177
  • DOI: 10.1038/srep18177
  • NAID: 120005712940
  • Peer Reviewed / Open Access
• [Journal Article] Construction and Characterization of Cy3- or Cy5-Conjugated Hairpin Pyrrole / Imidazole Polyamide Binding to DNA in the Nucleosome (2014)
  • Author(s): Han, Y-W.; Tsunaka, Y.; Yokota, H.; Matsumoto, T.; Kashiwazaki, G.; Morinaga, H.; Hashiya, K.; Bando, T.; Sugiyama, H.; Harada, Y.
  • Journal Title: Biomater. Sci. Volume: 2 Issue: 3 Pages: 297-307
  • DOI: 10.1039/c3bm60202h
  • NAID: 120005587835
  • Peer Reviewed
• [Journal Article] Solid-Supported DNA for Asymmetric Synthesis: a Stepping-Stone toward Practical Applications (2013)
  • Author(s): Han, Y-W.; Kashiwazaki, G.; Morinaga, H.; Matsumoto, T.; Hashiya, K.; Bando, T.; Sugiyama, H.
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 17 Pages: 5436-5441
  • DOI: 10.1016/j.bmc.2013.06.005
  • Peer Reviewed
• [Journal Article] In vivo fluorescent ATP imaging of Drosophila melanogaster and Caenorhabditis elegans by using a genetically encoded fluorescent ATP biosensor optimized for low temperatures. (2013)
  • Author(s): Tsuyama, Taiichi; Kishikawa, Jun-ichi; Han, Yong-Woon; Harada, Yoshie; Tsubouchi, Asako; Noji, Hiroyuki; Kakizuka, Akira; Yokoyama, Ken; Uemura, Tadashi; Imamura, Hiromi.
  • Journal Title: Analytical Chemistry Volume: 85(16) Issue: 16 Pages: 7889-7896
  • DOI: 10.1021/ac4015325
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition (2013)
  • Author(s): Naoko Mizuno, Marija Dramicanin, Michiyo Mizuuchi, Julia Adam, Yi Wang, Yong-Woon Han Wei Yang, Alasdair C. Steven, Kiyoshi Mizuuchi and Santiago Ramon-Maiques
  • Journal Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 110 Issue: 27
  • DOI: 10.1073/pnas.1309499110
  • Peer Reviewed
• [Journal Article] ParA-mediated plasmid partition driven by protein pattern self-organization (2013)
  • Author(s): Ling-Chin Hwang、Anthony G. Vecchiarelli、Yong-Woon Han、Michiyo Mizuuchi、Yoshie Harada、Barbara E. Funnell and Kiyoshi Mizuuchi
  • Journal Title: The EMBO Journal Volume: 32巻 Issue: 9 Pages: 1238-1249
  • DOI: 10.1038/emboj.2013.34
  • Peer Reviewed
• [Presentation] Biochemical analysis of SRA, hemi-methylated CpG binding domain (2016)
  • Author(s): Yong-Woon Han
  • Organizer: 60th annual meeting of biophysical society
  • Place of Presentation: ロスアンゼルス、米国
  • Year and Date: 2016-02-27
  • Int'l Joint Research
• [Presentation] RuvA-RuvB-Hollliday構造DNA複合体形成におけるATPの影響 (2016)
  • Author(s): 韓 龍雲
  • Organizer: 2016年度生体運動研究合同班会議
  • Place of Presentation: キャンパスプラザ京都（京都府、京都市）
  • Year and Date: 2016-01-08
• [Presentation] メチル化CpG維持に関与するSRAドメインの機能解析 (2015)
  • Author(s): 韓 龍雲
  • Organizer: 第38回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場（兵庫県、神戸市）
  • Year and Date: 2015-12-01
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation using Zero mode waveguides (2015)
  • Author(s): Yong-Woon Han
  • Organizer: iCeMS International Symposium, Hierarchical Dynamics in Soft Materials and Biological Matter
  • Place of Presentation: 京都大学（京都府、京都市）
  • Year and Date: 2015-09-23
  • Int'l Joint Research
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation using Zero Mode Waveguides (2015)
  • Author(s): Yong-Woon Han
  • Organizer: 第53回日本生物物理学会
  • Place of Presentation: 金沢大学（石川県、金沢市）
  • Year and Date: 2015-09-13
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation process using Single molecule fluorescence imaging technique (2015)
  • Author(s): Yong-Woon Han
  • Organizer: The 18th iCeMS International Symposium
  • Place of Presentation: 京都府、京都市
  • Year and Date: 2015-03-02 – 2015-03-04
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation process (2015)
  • Author(s): Yong-Woon Han
  • Organizer: 59th annual meeting of biophysical society
  • Place of Presentation: ボルチモア、米国
  • Year and Date: 2015-02-07 – 2015-02-11
• [Presentation] クロマチンリモデリング因子Paf complexの機能解析 (2015)
  • Author(s): 韓 龍雲
  • Organizer: ２０１５年度生体運動研究合同班会議
  • Place of Presentation: 東京、豊島区
  • Year and Date: 2015-01-07 – 2015-01-09
• [Presentation] Characterization of RuvB complex formation with a RuvA-Holliday junction DNA using Zero Mode Waveguides (2014)
  • Author(s): Yong-Woon Han
  • Organizer: 3R Symposium
  • Place of Presentation: 静岡県、三島市
  • Year and Date: 2014-11-17 – 2014-11-21
• [Presentation] Characterization of ATP-dependent chromatin remodeling complexes using fluorescently labeled nucleosome (2014)
  • Author(s): Yong-Woon Han
  • Organizer: 第５２回日本生物物理学会年会
  • Place of Presentation: 北海道、札幌市
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Characterization of RuvA-RuvB-Holliday junction DNA complex formation process using Zero Mode Waveguides (2014)
  • Author(s): Yong-Woon Han
  • Organizer: Graduate School of Biostudies & iCeMS Joint Symposium
  • Place of Presentation: 京都府、京都市
  • Year and Date: 2014-09-22
  • Invited
• [Presentation] ヌクレオソーム上のDNAに結合するCy3-又はCy5-標識Pyrrole/Imidazoleポリアミドの合成と機能解析 (2014)
  • Author(s): 韓 龍雲
  • Organizer: 第８回エピジェネティクス研究会年会
  • Place of Presentation: 東京都、文京区
  • Year and Date: 2014-05-25 – 2014-05-27
• [Presentation] Construction and characterization of Cy3- or Cy5-conjugated hairpin Pyrrole/Imidazole polyamides binding to DNA in the nucleosome (2014)
  • Author(s): Yong-Woon Han
  • Organizer: Biophysical Society 58th Annual Meeting
  • Place of Presentation: サンフランシスコ、米国
• [Presentation] 相同組換え中間体Holliday構造DNAの分岐点移動を促進するRuvB６量体リング構造の安定性に関する研究 (2014)
  • Author(s): 韓 龍雲
  • Organizer: ２０１４年度生体運動合同班会議
  • Place of Presentation: 千葉県、千葉市
• [Presentation] ヘミメチルCpG認識に関与するSRAドメインの機能解析 (2013)
  • Author(s): 韓 龍雲
  • Organizer: 第３６回日本分子生物学会年会
  • Place of Presentation: 兵庫県、神戸市
• [Presentation] Characterization of SRA-DNA complex using Zero Mode Waveguides (2013)
  • Author(s): Yong-Woon Han
  • Organizer: 第５１回日本生物物理学会年会
  • Place of Presentation: 京都府、京都市
• [Presentation] 一分子計測法によるSRA-DNA複合体の機能解析 (2013)
  • Author(s): 韓 龍雲
  • Organizer: 第７回日本エピジェネティクス研究会年会
  • Place of Presentation: 奈良県、奈良市