A novel technique producing hybrid proteins by using homologous recombination in vitro

2011 Fiscal Year Final Research Report

• Project/Area Number: 21200058
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research a proposed research project)
• Allocation Type: Single-year Grants
• Research Field: Applied biochemistry; Chemistry related to living body
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: MIKAWA Tsutomu 独立行政法人理化学研究所, 遺伝制御科学特別研究ユニット, 専任研究員 (20321820)
• Project Period (FY): 2009 – 2011
• Keywords: バイオテクノロジー / タンパク質工学 / タンパク質

Research Abstract

The aim of this study is to reconstitute homologous recombination in vitro, which can be used to produce hybrid genes and proteins. In this study, all proteins which participate in homologous recombination were prepared. Using these proteins, each step of homologous recombination was reconstituted in vitro. After optimization of each step, whole homologous recombination system was successfully reconstituted in vitro.

Research Products

• [Journal Article] The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay (2012)
  • Author(s): Yoshinori Shingu, Takeshi Tokai, Yasuo Agawa, Kentaro Toyota, Selina Ahamed, Makiko Kawagishi-Kobayashi, Akira Komatsu, Tsutomu Mikawa, Masa-Toshi Yamamoto, Kyo Wakasa, Takehiko Shibata, Kohji Kusano
  • Journal Title: BMC molecular biology Volume: vol.13 Pages: 1
  • Peer Reviewed
• [Journal Article] Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination (2011)
  • Author(s): Naoto Arai, Wataru Kagawa, Kengo Saito, Yoshinori Shingu, Tsutomu Mikawa, Hitoshi Kurumizaka, Takehiko Shibata
  • Journal Title: Journal of Biological Chemistry Volume: Vol.286, No.20 Pages: 17607-17617
  • DOI: 10.1074/jbc.M110.216739
  • Peer Reviewed
• [Journal Article] A mechanism for SSB displacement from single-stranded DNA upon SSB-RecO interaction (2011)
  • Author(s): Jin Inoue, Takayuki Nagae, Masaki Mishima, Yutaka Ito, Takehiko Shibata, Tsutomu Mikawa
  • Journal Title: Journal of Biological Chemistry Volume: Vol.286, No.8 Pages: 6720-6732
  • Peer Reviewed
• [Journal Article] Enlightenment of yeast mitochondrial homoplasmy: diversified role of gene conversion (2011)
  • Author(s): Feng Ling, Tsutomu Mikawa, Takehiko Shibata
  • Journal Title: Genes Volume: Vol.2 Pages: 169-190
  • Peer Reviewed
• [Journal Article] NMR protein structure determination in living E. coli cells using non-linear sampling (2011)
  • Author(s): Teppei Ikeya, Atsuko Sasaki, Daisuke Sakakibara, Yoshiki Shigemitsu, Junpei Hamatsu, Tomomi Hanashima, Masaki Mishima, Masatoshi Yoshimasu, Nobuhiro Hayashi, Tsutomu Mikawa, Daniel Nietlispach, Markus Walchli, Brian O. Smith, Masahiro Shirakawa, Peter Guntert, Yutaka Ito
  • Journal Title: Nature Protocols Volume: Vol.5, No.6 Pages: 1051-1060
  • Peer Reviewed
• [Journal Article] A DNA- binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis (2010)
  • Author(s): Yoshinori Shingu, Tsutomu Mikawa, Mariko Onuma, Takashi Hirayama, Takehiko Shibata
  • Journal Title: FEBS journal Volume: Vol.277, No.10 Pages: 2360-2374
  • Peer Reviewed
• [Journal Article] 相同DNA組換えの機構理解から新規蛋白質創出へ (2010)
  • Author(s): 柴田武彦, 美川務
  • Journal Title: 酵素工学ニュース Volume: Vol.63 Pages: 16-22
  • Peer Reviewed
• [Journal Article] Analysis of DNA-binding sites on Mhr1p, a yeast mitochondrial ATP-independent homologous pairing protein (2010)
  • Author(s): Tokiha Masuda, Feng Ling , Takehiko Shibata, Tsutomu Mikawa
  • Journal Title: FEBS journal Volume: Vol.277, No.6 Pages: 1440-1452
  • Peer Reviewed
• [Journal Article] A non-canonical DNA structure enables homologous recombination in various genetic systems (2009)
  • Author(s): Tokiha Masuda, Yutaka Ito, Tohru Terada, Takehiko Shibata, Tsutomu Mikawa
  • Journal Title: Journal of Biological Chemistry Volume: Vol.284, No.44 Pages: 30230-30239
  • Peer Reviewed
• [Journal Article] Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification (2009)
  • Author(s): Tsutomu Mikawa, Jin Inoue, Yasushi Shigemori
  • Journal Title: Analytical biochemistry Volume: Vol.391, No.2 Pages: 81-84
  • Peer Reviewed
• [Journal Article] DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange (2009)
  • Author(s): Takako Ishida, Yoshimasa Takizawa, Takashi Kainuma, Jin Inoue, Tsutomu Mikawa, Takehiko Shibata, Hidekazu Suzuki, Satoshi Tashiro, Hitoshi Kurumizaka
  • Journal Title: Nucleic Acids Research Volume: Vol.37, No.10 Pages: 3367-3376
  • Peer Reviewed
• [Journal Article] RecA-based PCRの開発と応用 (2009)
  • Author(s): 美川務
  • Journal Title: 月刊バイオインダストリー Volume: Vol.26 Pages: 58-64
  • Peer Reviewed
• [Journal Article] Epitope mapping by region-specified PCR-mutagenesis (2009)
  • Author(s): Tsutomu Mikawa, Masayuki lkedaand Takehiko Shibata
  • Journal Title: Methods in Molecular Biology Volume: Vol.524 Pages: 305-313
  • Peer Reviewed
• [Presentation] Reconstitution of homologous recombination in vitro by using Thermus thermophilus RecF pathway proteins (2012)
  • Author(s): 井上仁
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-16
• [Presentation] Mitochondrial DNA homoplasmy as gene homogenization of repeated sequences by rolling circle replication (2012)
  • Author(s): 柴田武彦
  • Organizer: 7th ASMRM and 10th J-mit
  • Place of Presentation: 福岡県福岡市
  • Year and Date: 2012-12-16
• [Presentation] A novel mechanism for the inhibition of RecA filament extension by RecX (2012)
  • Author(s): 奥居沙弥
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-15
• [Presentation] Analysis of the functions of recombination mediators in homologous pairing (2012)
  • Author(s): 篠原赳
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-15
• [Presentation] 出芽酵母Rad52-Rad51によるD-loop形成におけるRad52のC端ドメインの必要性 (2012)
  • Author(s): 新井直人
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-14
• [Presentation] Local folding of the N-terminal domain of RecA upon protein-protein / protein-DNA interactions and its role (2012)
  • Author(s): 美川務
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-14
• [Presentation] 酵素結晶固定化電極を用いたバイオ電池の高出力化 (2012)
  • Author(s): 重森康司
  • Organizer: 第33回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2012-12-13
• [Presentation] バクテリアにおけるDNA組換え修復系 (2011)
  • Author(s): 井上仁
  • Organizer: 第21回DNA複製・組換え・ゲノム安定性制御ワークショップ
  • Place of Presentation: 福岡県福津市
  • Year and Date: 2011-10-25
• [Presentation] 天然変性領域としてのRecAのN末端ドメイン:その機能と役割 (2011)
  • Author(s): 美川務
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都府京都市
  • Year and Date: 2011-09-23
• [Presentation] 高度好熱菌蛋白質を用いたin vitro DNA相同組換えの再構築 (2011)
  • Author(s): 井上仁
  • Organizer: 第1回モデル生物丸ごと一匹学会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2011-08-20
• [Presentation] RecAの組換え反応に対するRecXの阻害機構 (2011)
  • Author(s): 奥居沙弥
  • Organizer: 第1回モデル生物丸ごと一匹学会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2011-08-19
• [Presentation] 組換えメディエーターによる D-loop 反応制御 (2011)
  • Author(s): 篠原赳
  • Organizer: 第1回モデル生物丸ごと一匹学会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2011-08-19
• [Presentation] 蛋白質間相互作用により生じる単鎖DNA結合蛋白質（SSB/RPA）のssDNAからの解離の一般的機構 (2011)
  • Author(s): 井上仁
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: 大阪府吹田市
  • Year and Date: 2011-06-08
• [Presentation] Displacement mechanism for SSB from single-stranded DNA by a recombination mediator: SSB-RecO interaction at the atomic resolution (2011)
  • Author(s): 井上仁
  • Organizer: International Symposium on the Physicochemical Field for Genetic Activities
  • Place of Presentation: 兵庫県淡路市
  • Year and Date: 2011-01-24
• [Presentation] 出芽酵母Rad51によるD-loop形成促進のためのRad52第二DNA結合部位の機能 (2011)
  • Author(s): 新井直人
  • Organizer: 第28回染色体ワークショップ
  • Place of Presentation: 石川県加賀市
  • Year and Date: 2011-01-12
• [Presentation] ssDNA上でのSSBの置換におけるSSB-蛋白質相互作用の役割 (2010)
  • Author(s): 井上仁
  • Organizer: 第33回日本分子生物学会年会・第83回日本生化学会大会 (BMB2010)
  • Place of Presentation: 兵庫県神戸市
  • Year and Date: 2010-12-09
• [Presentation] RecA/Rad51ファミリーのフィラメント形成は相同DNA対合反応には必要ない (2010)
  • Author(s): 増田ときは
  • Organizer: 第33回日本分子生物学会年会・第83回日本生化学会大会 (BMB2010)
  • Place of Presentation: 兵庫県神戸市
  • Year and Date: 2010-12-07
• [Presentation] 出芽酵母Rad52の第二DNA結合部位も相同組換えに必要である (2010)
  • Author(s): 新井直人
  • Organizer: 第33回日本分子生物学会年会・第83回日本生化学会大会 (BMB2010)
  • Place of Presentation: 兵庫県神戸市
  • Year and Date: 2010-12-07
• [Presentation] A mechanism for SSB displacement from single-stranded DNA upon SSB-protein interaciton (2010)
  • Author(s): 井上仁
  • Organizer: The 7^<th> International 3R Symposium
  • Place of Presentation: 富山県富山市
  • Year and Date: 2010-10-27
• [Presentation] A mechanism of DNA homology search, based on the structure of DNA bound to proteins that promote homologous recombination (2010)
  • Author(s): Tokiha Masuda
  • Organizer: The 12^<th> IUBMB (OzBio2010)
  • Place of Presentation: Melbourne, Australia
  • Year and Date: 2010-09-27
• [Presentation] A general mechanism for SSB displacement from ssDNA upon SSB-protein interaction (2010)
  • Author(s): 井上仁
  • Organizer: 「高度好熱菌丸ごと一匹プロジェクト」第9回連携研究会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2010-08-20
• [Presentation] Direct interaction of RecO with RecA is involved in regulation of RecA function (2010)
  • Author(s): 篠原赳
  • Organizer: 「高度好熱菌丸ごと一匹プロジェクト」第9回連携研究会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2010-08-20
• [Presentation] Analysis of the inhibition mechanism of extension of RecA filament by RecX (2010)
  • Author(s): 奥居沙弥
  • Organizer: 「高度好熱菌丸ごと一匹プロジェクト」第9回連携研究会
  • Place of Presentation: 兵庫県佐用郡
  • Year and Date: 2010-08-20
• [Presentation] The universal DNA structure in homologous recombination (2010)
  • Author(s): Tsutomu Mikawa
  • Organizer: The24th Symposium of the Protein Society
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2010-08-03
• [Presentation] 相同組換え開始制御に働く天然変性タンパク質の機能 (2010)
  • Author(s): 柴田武彦
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2010-06-18
• [Presentation] SSB-C末端酸性領域とRecOの相互作用によるSSBのssDNAからの解離機構の解明 (2010)
  • Author(s): 井上仁
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2010-06-17
• [Presentation] RecAはなぜフィラメントを形成する必要があるのか? -単量体及び二量体の活性をもとに- (2010)
  • Author(s): 笠置原央
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2010-06-17
• [Presentation] A novel rice SPO11homologue, SPO11D, induces meiotic chromosome aberration and DNA double-stranded breaks in the fruit-fly (2010)
  • Author(s): Yoshinori Shingu
  • Organizer: EMBO Conference Series on Recombination & Connections to SUMO and Ubiquitin Modifications
  • Place of Presentation: Lucca, Italy
  • Year and Date: 2010-05-19
• [Presentation] 相同DNA組換えにおける2種の相同DNA対合蛋白質の役割分担 (2010)
  • Author(s): 柴田武彦
  • Organizer: 第27回染色体ワークショップ
  • Place of Presentation: 静岡県御殿場市
  • Year and Date: 2010-01-22
• [Presentation] イネの減数分裂組換え開始酵素「Spo11」 (2010)
  • Author(s): 新宮良宣
  • Organizer: 第27回染色体ワークショップ
  • Place of Presentation: 静岡県御殿場市
  • Year and Date: 2010-01-22
• [Presentation] The universal structure of ssDNA in homologous recombination (2009)
  • Author(s): 増田ときは
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2009-12-11
• [Presentation] RecAに対するRecXの阻害効果は種特異的である (2009)
  • Author(s): 奥居沙弥生
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2009-12-11
• [Presentation] NMR analyses of interactions of RecO with SSB and ssDNA (2009)
  • Author(s): 井上仁
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2009-12-11
• [Presentation] RecAの最小機能単位 -単量体及び二量体の活性- (2009)
  • Author(s): 笠置原央
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2009-12-11
• [Presentation] 出芽酵母Rad52-Rad51複合体によるD-loop形成においてRad52は二本鎖DNAの結合に働く (2009)
  • Author(s): 新井直人
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 神奈川県横浜市
  • Year and Date: 2009-12-11
• [Presentation] 相同DNA組換えにおける天然変性領域の機能 (2009)
  • Author(s): 柴田武彦
  • Organizer: 第82回日本生化学会大会
  • Place of Presentation: 兵庫県神戸市
  • Year and Date: 2009-10-21
• [Presentation] Thermus thermophilusHB8のDNA相同組換え系 (2009)
  • Author(s): 美川務
  • Organizer: 「高度好熱菌丸ごと一匹プロジェクト」第8回連携研究会
  • Place of Presentation: 兵庫県佐用郡佐用町
  • Year and Date: 2009-08-22
• [Presentation] Identification of the SSB binding site on RecO by NMR analysis (2009)
  • Author(s): 井上仁
  • Organizer: 「高度好熱菌丸ごと一匹プロジェクト」第8回連携研究会
  • Place of Presentation: 兵庫県佐用郡佐用町
  • Year and Date: 2009-08-22
• [Patent(Industrial Property Rights)] 酵素結晶固定化電極及び酵素結晶固定化電極の製造方法、並びにこれを備えるバイオ燃料電池及びバイオセンサー (2011)
  • Inventor(s): 重森康司, 美川務
  • Industrial Property Rights Holder: (独)理化学研究所, アイシン精機(株)
  • Industrial Property Number: 国際特許PCT/JP2011/069060
  • Filing Date: 2011-08-24
• [Patent(Industrial Property Rights)] 高度好熱菌由来の改変型一本鎖DNA結合タンパク質、及び該タンパク質を用いた核酸の等温増幅方法 (2011)
  • Inventor(s): 美川務, 柴田武彦. 重森康司
  • Industrial Property Rights Holder: (独)理化学研究所, アイシン精機(株)
  • Industrial Property Number: 特許,特願486099
  • Acquisition Date: 2011-11-11
  • Overseas
• [Patent(Industrial Property Rights)] 高度好熱菌由来の改変型一本鎖DNA結合タンパク質、及び該タンパク質を用いた核酸の等温増幅方法 (2011)
  • Inventor(s): 美川務, 柴田武彦. 重森康司
  • Industrial Property Rights Holder: (独)理化学研究所, アイシン精機(株)
  • Industrial Property Number: 特許,US Patent7973131
  • Acquisition Date: 2011-07-05
• [Patent(Industrial Property Rights)] 酵素結晶固定化電極及び酵素結晶固定化電極の製造方法、並びにこれを備えるバイオ燃料電池及びバイオセンサー (2010)
  • Inventor(s): 重森康司, 美川務
  • Industrial Property Rights Holder: (独)理化学研究所, アイシン精機(株)
  • Industrial Property Number: 特許,特願2010-189788
  • Filing Date: 2010-08-26
  • Overseas