Method to enhance gene-targeting efficiency

• Project/Area Number: 20770147
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Kyoto University
• Principal Investigator: HIROTA Kouji Kyoto University, 大学院・医学研究科, 助教 (00342840)
• Project Period (FY): 2008 – 2009
• Project Status: Completed (Fiscal Year 2009)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2009: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2008: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 相同組換え / 標的組換え / DT40 / Blm / Exo1 / 組換え促進法 / トリBリンパ球 / 組換え / DT40細胞 / 細胞分化 / transcriptome

Research Abstract

Bloom syndrome is caused by inactivation of the Bloom DNA helicase(Blm). To investigate the role of Blm in gene targeting event, we analyzed the chicken DT40 B lymphocyte line. We measured the frequency of gene-targeting induced by an I-Sce1-endonuclease-mediated double-strand break (DSB). BLM-/- cells showed a severer defect in the gene-targeting frequency, as the number of heterologous sequences increased at the DSB site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene-targeting. Moreover we discovered that Exo1, exonuclease also activates this gene-targeting event collaborating with Blm helicase. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.

Research Products

• [Journal Article] Analysis of chromatin Fukuda, T., Yamada, T. and structure at meiotic DSB sites in yeasts. (2009)
  • Author(s): Hirota, K., Ohta, K.
  • Journal Title: Methods in molecular biology Clifton, N. J 557 Pages: 253-266
  • Peer Reviewed
• [Journal Article] helicase facilitates homologous Bloom DNA recombination between diverged homologous sequences. (2009)
  • Author(s): Kikuchi, K., Abdel-Aziz, H.I., Taniguchi, Y., Yamazoe, M., Takeda, S., Hirota, K.
  • Journal Title: The Journal of biological chemistry 284 Pages: 26360-26367
  • Peer Reviewed
• [Journal Article] transcription of mRNA-type long and Ohta, K. Cascade non-coding RNAs (mlonRNAs) and local chromatin remodeling. (2009)
  • Author(s): Hirota, K.
  • Journal Title: Epigenetics 4 Pages: 5-7
  • Peer Reviewed
• [Journal Article] Transcription of mRNA-type long and Ohta, K. non-coding RNAs (mlonRNAs) disrupts chromatin array. (2009)
  • Author(s): Hirota, K.
  • Journal Title: Communicative & integrative biology 2 Pages: 25-26
  • Peer Reviewed
• [Journal Article] Analysis of chromatin structure at meiotic DSB sites in yeasts. (2009)
  • Author(s): Hirota, K., Fukuda, T., Yamada, T., Ohta, K.
  • Journal Title: Methods in molecular biology Clifton, N.J VOL.557 Pages: 253-266
• [Journal Article] Bloom DNA helicase facilitates homologous recombination between diverged homologous sequences. (2009)
  • Author(s): Kikuchi, K., Abdel-Aziz, H.I., Taniguchi, Y., Yamazoe, M., Takeda, S., Hirota, K.
  • Journal Title: The Journal of biological chemistry VOL.284 Pages: 26360-26367
  • Peer Reviewed
• [Journal Article] Cascade transcription of mRNA-type long non-coding RNAs (mlonRNAs) and local chromatin remodeling (2009)
  • Author(s): Hirota, K., and Ohta, K
  • Journal Title: Epigenetics 4 Pages: 5-7
  • Peer Reviewed
• [Journal Article] Transcription of mRNA-type long non-coding RNAs (mlonRNAs) disrupts chromatin array (2009)
  • Author(s): Hirota, K., and Ohta, K.
  • Journal Title: Communicative & Integrative Biology 2 Pages: 25-26
  • Peer Reviewed
• [Journal Article] Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs. (2008)
  • Author(s): Hirota, K. Hoffman, C.S., Shibata, T., Ohta, K., Miyoshi, T., Kugou, K.
  • Journal Title: Nature 456 Pages: 130-134
  • Peer Reviewed
• [Journal Article] In-cell Viscosity, Ohta, K. and Measurement Using a Fluorescence Up-conversion Microscope. (2008)
  • Author(s): Fujino, T., Hirota, K. Tahara, T.
  • Journal Title: Chemistry Letters 37 Pages: 1240-1240
  • Peer Reviewed
• [Journal Article] Distinct chromatin, Mizuno, K., Shibata, T. and modulators regulate the formation of accessible and repressive chromatin at the fission yeast recombination hotspot ade6-M26. (2008)
  • Author(s): Hirota, K. Ohta, K.
  • Journal Title: Molecular biology of the cell 19 Pages: 1162-1173
  • Peer Reviewed
• [Journal Article] Cdc7-dependent phosphorylation of Mer2 facilitates initiation of yeast meiotic recombination. (2008)
  • Author(s): Sasanuma, H., Hirota, K., Kakusho, N., Kugou, K., Kawasaki, Y., Fukuda, T., Shibata, T., Masai, H., Ohta, K.
  • Journal Title: Genes & development 22 Pages: 398-410
  • Peer Reviewed
• [Journal Article] Essential roles of Snf21, a Swi2/Snf2 family chromatin remodeler, in fission yeast mitosis. (2008)
  • Author(s): Yamada, K., Hirota, K., Shibata, T., Ohta, K. Mizuno, K.
  • Journal Title: Genes & genetic systems 83 Pages: 361-372
  • NAID: 10023887824
  • Peer Reviewed
• [Journal Article] Structural and functional analyses of the DMC1-M200V polymorphism found in the human population. (2008)
  • Author(s): Hikiba, J., Hirota, K., Ikawa, S., Kinebuchi, T., Sakane, I., Kagawa, W., Takizawa, Y., Yokoyama, S., Mandon-Pepin, B., Nicolas, A., et al.
  • Journal Title: Nucleic acids research 36 Pages: 4181-4190
  • Peer Reviewed
• [Journal Article] Structural and functional analyses of the DMC1-M200V Polymorphism found in the human population (2008)
  • Author(s): Hikiba, J., Hirota, K. Kagawa. B., Ikara, S., Kinebuchi, T., Sakane, I., Takizawa, Y. Yokoyams, S., Mandan-Pepin, B. Nicolas, A., Kurumizake II.
  • Journal Title: Nucleic acids research 36 Pages: 4181-4190
  • Peer Reviewed
• [Journal Article] Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs (2008)
  • Author(s): Hirota, K.,Miyoshi, T., Kugou, K., Hoffman, C.S., Shibata, T., and Ohta, K.
  • Journal Title: Nature 456 Pages: 130-134
  • Peer Reviewed
• [Journal Article] Essential roles of Snf21, a Swi2/Snf2 family chromatin remodeler, in fission yeast mitosis (2008)
  • Author(s): Yamada, K., Hirota, K.,, Mizuno, K., Shibata, T., and Ohta, K.
  • Journal Title: Genes & genetic systems 83 Pages: 361-372
  • NAID: 10023887824
  • Peer Reviewed
• [Presentation] Chromatin regulation of meiotic recombination and stress-induced gene activation. (2009)
  • Author(s): Miyoshi, T., Kugou, K., Ohta, K. Koji Kikuchi, H. Ismail Abdel-Aziz, Mitsuyoshi Yamazoe, Kouji Hirota
  • Organizer: International Symposium DNA, Miyoshi
  • Place of Presentation: Damage Response and Repair
• [Presentation] AGENDA GeneExpression Systems of USA Shunichi Takeda The Bloom DNA helicase facilitates homologous recombination between diverged sequences Presents Epigenetics (2009)
  • Author(s): Kouji Hirota
  • Organizer: Sequencing &SNiPs-2009 Meeting
• [Presentation] Non coding RNAs disrupt chromatin array Transcription of mRNA-type long non-coding RNAs (mlonRNAs) disrupts chromatin array (2009)
  • Author(s): Kouji Hirota
  • Organizer: 内藤コンファレンスNuclear Dynamics and RNA
• [Presentation] The Bloom DNA helicase facilitates homologous recombination between diverged sequences (2009)
  • Author(s): Koji Kikuchi, H.Ismail Abdel-Aziz, Mitsuyoshi Yamazoe, Kouji Hirota, Shunichi Takeda
  • Organizer: International Symposium DNA Damage Response and Repair Mechanisms
  • Place of Presentation: ギリシャ(クレタ)
• [Presentation] Non coding RNAs disrupt chromatin array (2009)
  • Author(s): Kouji Hirota
  • Organizer: AGENDA GeneExpression Systems of USA Presents Epigenetics, Sequencing &SNiPs-2009 Meeting
  • Place of Presentation: 米国(ハーバード大学)
• [Presentation] Transcription of mRNA-type long non-coding RNAs (mlonRNAs) disrupts chromatin array (2009)
  • Author(s): Kouji Hirota
  • Organizer: 内藤コンファレンスNuclear Dynamics and RNA
  • Place of Presentation: 日本(北海道)
• [Presentation] (2008)
  • Author(s): Hirota, K.
  • Organizer: 分子生物学会(BMB2008)
  • Year and Date: 2008-12-12
• [Presentation] Chromatin regulation of meiotic recombination and stress-induced gene activation (2008)
  • Author(s): Hirota, K., Miyoshi, T., Kugou, K., Ohta, K.
  • Organizer: 分子生物学会(BMB2008)
  • Place of Presentation: 神戸
  • Year and Date: 2008-12-12
• [Book] 細胞工学(基礎の基礎, 1月号) (2010)
  • Author(s): 廣田耕志、武田俊一
• [Book] 化学と生物(mlonRNA仮説ノンコーディングRNA転写による新規クロマチン構造変化機構, 5月号) (2009)
  • Author(s): 廣田耕志、太田邦史
• [Book] RNA蛋白質核酸酵素(クロマチン再編成をつうじて遺伝子活性化にかかわるmRNA型長鎖ノンコーディング, No6 vol54) (2009)
  • Author(s): 廣田耕志、太田邦史
• [Book] 細胞工学12月号(基礎の基礎) (2009)
  • Author(s): 廣田耕志、武田俊一
  • Total Pages: 7
  • Publisher: 学研メディカル秀潤社
• [Book] タンパク質核酸酵素 (2009)
  • Author(s): 廣田耕志、太田邦史
  • Publisher: 共立出版
• [Book] 化学と生物 (2009)
  • Author(s): 廣田耕志、太田邦史
  • Publisher: 日本農芸化学会
• [Remarks]
  • URL: http://rg4.rg.med.kyoto-u.ac.jp/
• [Patent(Industrial Property Rights)] 遺伝的に改変された細胞を製造する方法 (2009)
  • Inventor(s): 武田俊一, 廣田耕志
  • Industrial Property Rights Holder: 独立行政法人農業生物資源研究所
  • Industrial Property Number: 2009-138902
  • Filing Date: 2009-06-10