Stabilization of stalled replication forks by fork-binding proteins

• Project/Area Number: 23657081
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: MASAI Hisao 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 基盤技術研究センター長 (40229349)
• Co-Investigator(Kenkyū-buntansha): TANAKA Taku 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 主席研究員 (80425686)
• Co-Investigator(Renkei-kenkyūsha): MATUMOTO Seiji 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 研究員 (40190532) ; KAKUSHO Naoko 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 研究員 (30599593) ; KANOH Yutaka 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 研究員 (90450593)
• Project Period (FY): 2011 – 2012
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2011: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: PriAタンパク質 / 複製フォーク / 複製ストレス / RecGタンパク質 / チェックポイント応答 / 染色体脆弱部位 / DNA結合タンパク質 / 染色体不安定性症候群 / 複製複合体 / DNA複製停止 / ゲノム不安定性

Research Abstract

We previously showed that E. coli PriA protein, in collaboration with RecG, specifically binds and can stabilize an arrested replication fork. In this proposal we attempted to stabilize stalled replication forks in eukaryotic cells by utilizing these two proteins. We first developed an antibody against PriA and showed that stalled replication forks can be detected by ChIP-chip analyses. In response to fork arrest treatment, PriA bound to oriC in the wild-type cells, whereas it bound to the terC region under the condition where oriC is not functional. We then expressed PriA in fission yeast cells, examined its binding to chromatin. PriA was enriched near the replication origins. We are now expressing PriA in various mutant cells, and will examine if PriA can stabilize the fork by measuring the length of SSB coated single-strand.

Research Products

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• [Journal Article] Cell cycle synchronization and flow cytometry analysis of mammalian cell (2014)
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• [Journal Article] News and Views "ATM in prevention of genomic instability" (2014)
  • Author(s): Hisao Masai
  • Journal Title: Cell Cycle Volume: 13 Pages: 882-883
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• [Journal Article] "Temporal and spatial regulation of eukaryotic DNA replication: From regulated initiation to genome-scale timing program." (2014)
  • Author(s): Renard-Guillet, C., Kanoh, Y., Shirahige, K., and Masai, H.
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• [Journal Article] Regulation and roles of Cdc7 kinase under replication stress (2014)
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  • Journal Title: Cell Cycle May Volume: 19
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• [Journal Article] "Cell cycle synchronization and flow cytometry analysis of mammalian cell." (2014)
  • Author(s): Yoshizawa-Sugata, N. and Masai, H
  • Journal Title: Methods in Molecular Biology Volume: in press
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• [Journal Article] "ATM in prevention of genomic instability." (2014)
  • Author(s): Hisao Masai
  • Journal Title: Cell Cycle Volume: 13 Pages: 882-883
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• [Journal Article] "Temporal and spatial regulation of eukaryotic DNA replication: From regulated initiation to genome-scale timing program." (2014)
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  • Journal Title: Seminars in Cell & Developmental Biology Volume: 24 Pages: 226-231
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• [Journal Article] "A personal reflection on the Replicon Theory: from R1 plasmid to replication timing regulation in human cells" (2013)
  • Author(s): Masai, H.
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  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Regulation of chromosome dynamics by Hsk1 kinase (2013)
  • Author(s): Matsumoto, S. and Masai, H
  • Journal Title: Biochemical Society Transactions Volume: 41 Pages: 1712-1719
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• [Journal Article] ATR-Chk1-APC/CCdh1-dependent stabilization of Cdc7-ASK(Dbf4) kinase complex is required for DNA damage bypass under replication stress (2013)
  • Author(s): Yamada, M., Watanabe, K., Mistrik, M., Mailand, N., Lee, M-H., Masai, H., Lukas, J. and Bartek, B
  • Journal Title: Genes and Development Volume: 27 Pages: 2459-2472
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• [Journal Article] "The Human Tim/Tipin Complex Directly Interacts with DNA Polymerase {epsilon} and Stimulates its Synthetic Activity." (2013)
  • Author(s): Aria, V., De Felice, M., Di Perna, R., Uno, S., Masai, H., Syvaoja, J.E., van Loon, B., Hubscher, U. and Pisani, F.M.
  • Journal Title: J. Biol. Chem Volume: 288 Pages: 12742-12752
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• [Journal Article] "Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation." (2013)
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  • Journal Title: Nucleic Acids Res Volume: 41 Pages: 8475-8488
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• [Journal Article] Mechanism of transition to invasive cancer, on basis of genomic instability and genetic alterations, and identification of a novel biomarker to predict poor prognosis in endometrial carcinomas (2013)
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  • Journal Title: Br. J. Cancer Volume: 109 Pages: 2388-2401
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• [Journal Article] Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob (2013)
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• [Journal Article] Replication timing regulation of eukaryotic replicons: Rif1 as a global regulator of replication timing (2013)
  • Author(s): Yamazaki, S. Hayano, M. and Masai, H.
  • Journal Title: Trends in Genetics. Volume: 29 Pages: 449-460
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• [Journal Article] The mini-chromosome maintenance (Mcm) complexes interact with DNA polymerase α-primase and stimulate its ability to synthesize RNA primers (2013)
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  • Journal Title: PLoS One Volume: 8
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• [Journal Article] "Regulation of chromosome dynamics by Hsk1 kinase." (2013)
  • Author(s): Matsumoto, S. and Masai, H
  • Journal Title: Biochemical Society Transactions Volume: 41 Pages: 1712-1719
  • Peer Reviewed
• [Journal Article] Replication timing regulation of eukaryotic replicons: Rif1 as a global regulator of replication timing. (2013)
  • Author(s): Yamazaki, S. Hayano, M. and Masai, H.
  • Journal Title: Trends in Genetics Volume: In press
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• [Journal Article] Rif1 is a global regulator of timing of replication origin firing in fission yeast. (2012)
  • Author(s): Hayano M, Kanoh Y, Matsumoto S, Renard-Guillet C, Shirahige K, Masai H
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  • DOI: 10.1101/gad.178491.111
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• [Journal Article] Rif1 protein is a key regulator of the genome-wide DNA replication timing in human cells (2012)
  • Author(s): Yamazaki, S., Ishii, A., Kanoh, Y., Oda, M., Nishito, Y. and Masai, H.
  • Journal Title: EMBO J Volume: 31 Pages: 3167-3177
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• [Journal Article] c-Jun N-terminal Kinase (JNK)-Mediated Rad18 Phosphorylation Facilitates Polη Recruitment to Stalled Replication Forks (2012)
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  • Journal Title: Mol. Biol. Cell Volume: 23 Pages: 1943-1954
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• [Journal Article] EBNA1-dependent recruitment of Orc on OriP of Epstein-Barr virus with purified proteins : Stimulation by Cdc6 through Its direct interaction with EBNA1 (2012)
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• [Journal Article] Purification of replication factors using insect and mammalian cell expression systems (2012)
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• [Journal Article] A Central Coupler for Recombination Initiation Linking Chromosome Architecture to S Phase Checkpoint. (2012)
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• [Journal Article] Cdc7l1 (2012)
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• [Journal Article] Cdc7 (2012)
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  • Journal Title: The Encyclopedia of Signaling Molecules Springer Reference and Database Publishing
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• [Journal Article] Dbf4 (2012)
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  • Journal Title: The Encyclopedia of Signaling Molecules Springer Reference and Database Publishing
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• [Journal Article] Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob (2012)
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• [Journal Article] Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1. (2012)
  • Author(s): Oda, M., Kanoh, Y., Watanabe, Y., and Masai, H.
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• [Journal Article] Mechanism of cancer cell death induced by depletion of an essential replication regulator. (2012)
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• [Journal Article] テロメア結合因子Rif1は、染色体複タイミングを決定する (2012)
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  • Journal Title: 実験医学、Current Topics Volume: 30 Pages: 2974-2978
• [Journal Article] Rif1 is a global regulator of timing of replication origin firing in fission yeast. (2012)
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• [Journal Article] RecQL4 : a helicase linking formation and maintenance of a replication fork (2011)
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• [Journal Article] Molecular mechanism of activation of human Cdc7 kinase : Bipartite interaction with Dbf4/ASK stimulates ATP binding and substrate recognition (2011)
  • Author(s): Kitamura, R., Fukatsu, R., Kakusho, N., Cho, Y-S., Taniyama, C., Yamazaki, S., Toh, G-T., Yanagi, K., Arai, N., Chang, H-J. and Masai, H.
  • Journal Title: J. Biol. Chem Volume: 286 Pages: 23031-23043
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• [Journal Article] Pre-firing binding of Mrc1 defines the early-firing origins which are selectively hyper-activated upon loss of fork stabilizing factors in fission yeast (2011)
  • Author(s): Hayano, M., Kanoh, Y., Matsumoto, S., Kakusho, N. and Masai, H.
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• [Journal Article] Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation (2011)
  • Author(s): Matsumoto, S., Hayano, M., Kanoh. Y. and Masai, H.
  • Journal Title: J. Cell Biol Volume: 195 Pages: 387-401
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• [Journal Article] Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation. (2011)
  • Author(s): Matsumoto S, Hayano M, Kanoh Y, Masai H.
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• [Journal Article] Mrc1 marks early-firing origins and coordinates timing and efficiency of initiation in fission yeast. (2011)
  • Author(s): Hayano M, Kanoh Y, Matsumoto S, Masai H.
  • Journal Title: Mol Cell Biol. Volume: 31 Pages: 2380-2391
  • Peer Reviewed
• [Presentation] 染色体DNA 複製プログラムの制御機構と起源・進化 (2014)
  • Author(s): 正井 久雄, 田中 卓, 加納 豊, 山崎 聡志, 松本 清治, 吉沢 直子, 松嶋 夢叶
  • Organizer: The 2nd Symposium of Cell Cycle Control and Cell Fate (第2回 細胞周期制御と細胞運命シンポジウム)
  • Place of Presentation: 浜松
  • Invited
• [Presentation] "染色体DNA複製プログラムの制御機構と起源・進化" (2014)
  • Author(s): 正井 久雄、田中 卓、加納 豊、山崎 聡志、松本 清治、吉沢 直子、松嶋 夢叶
  • Organizer: The 2nd Symposium of Cell Cycle Control and Cell Fate (第２回 細胞周期制御と細胞運命 シンポジウム)
  • Place of Presentation: 浜松
  • Invited
• [Presentation] Cell cycle and chromatin regulators in stem cell regulation (2013)
  • Author(s): Hisao Masai
  • Organizer: The Gene and Immnuotherapy conference
  • Place of Presentation: Ho chi Min City, Vietnam
  • Invited
• [Presentation] "A personal reflection on the Replicon Theory : from R1 plasmid to replication timing regulation in human cells" "Symposium : Half a Century With Replicon Theory for Genome Stability and Instability" (2013)
  • Author(s): Hisao Masai
  • Organizer: The 50th anniversary of the replicon theory
  • Place of Presentation: Pasteur Institute, Paris, France
  • Invited
• [Presentation] Control of origin firing timing in fission yeast cells (2013)
  • Author(s): Hisao Masai
  • Organizer: EMBO Conference "Pombe 2013 7th International Fission Yeast Meeting"
  • Place of Presentation: London UK
  • Invited
• [Presentation] "Introduction" Symposium (2013)
  • Author(s): Hisao Masai
  • Organizer: 86th Annual Meeting of the Japanese Biochemical Society
  • Place of Presentation: Fukuoka, Japan
  • Invited
• [Presentation] Chromatin architecture that regulates replication timing of eukaryotic chromosomes (2013)
  • Author(s): Hisao Masai, Yutaka Kanoh, Satoshi Yamazaki, Motoshi Hayano, Naoko Yoshizawa, Kenji Moriyama, Yumeka Matsushima, Naoko Kakusho, Rino Fukatsu
  • Organizer: 第86回日本生化学会大会事務局 インターナショナルセッション『Assembly and architecture of protein complexes regulating inheritance and stable maintenance of genome』
  • Place of Presentation: 福岡
  • Invited
• [Presentation] イントロダクション (2013)
  • Author(s): 正井 久雄
  • Organizer: 第36回(2013年)日本分子生物学会年会, ワークショップ 『レプリコン仮説50周年 : 染色体複製装置の形成とその活性の時空間制御』オーガナイザー
  • Place of Presentation: 福岡
  • Invited
• [Presentation] Renard-Guillet Claire "Rif1 : a potential global regulator of DNA replication, DNA repair, and recombination" (2013)
  • Author(s): 正井 久雄, 山崎 聡志, Lai Mongsing, Foiani Marco, 吉沢 直子, 加納 豊, 松本 清治, 森山賢治, 覺正 直子, 深津 理乃, 松嶋 夢叶, 河野 暢明
  • Organizer: 第36回(2013年)日本分子生物学会年会, ワークショップ 『DNA 二重鎖切断のend-resection と修復機構の選択・制御』
  • Place of Presentation: 福岡
  • Invited
• [Presentation] "Control of origin firing timing in fission yeast cells" (2013)
  • Author(s): Hisao Masai
  • Organizer: EMBO Conference "Pombe 2013: 7th International Fission Yeast Meeting"
  • Place of Presentation: London UK
  • Invited
• [Presentation] Introductory Remarks (2013)
  • Author(s): Hisao Masai
  • Organizer: 86th Annual Meeting of the Japanese Biochemical Society
  • Place of Presentation: 福岡
  • Invited
• [Presentation] “Chromatin architecture that regulates replication timing of eukaryotic chromosomes.“ (2013)
  • Author(s): Hisao Masai, Yutaka Kanoh, Satoshi Yamazaki, Motoshi Hayano, Naoko Yoshizawa, Kenji Moriyama, Yumeka Matsushima, Naoko Kakusho, and Rino Fukatsu
  • Organizer: 第86回日本生化学会大会事務局 インターナショナルセッション「Assembly and architecture of protein complexes regulating inheritance and stable maintenance of genome」
  • Place of Presentation: 福岡
  • Invited
• [Presentation] レプリコン仮説５０周年 (2013)
  • Author(s): 正井 久雄
  • Organizer: 第36回（2013年）日本分子生物学会年会，ワークショップ 「レプリコン仮説50周年：染色体複製装置の形成とその活性の時空間制御」
  • Place of Presentation: 福岡
  • Invited
• [Presentation] "Rif1: a potential global regulator of DNA replication, DNA repair, and recombination." (2013)
  • Author(s): 正井 久雄、山崎 聡志、Lai Mongsing、Foiani Marco、吉沢 直子、加納 豊、松本 清治、森山賢治、覺正 直子、深津 理乃、松嶋 夢叶、河野 暢明、Renard-Guillet Claire
  • Organizer: 第36回（2013年）日本分子生物学会年会，ワークショップ 「DNA二重鎖切断のend-resectionと修復機構の選択・制御」
  • Place of Presentation: 福岡
  • Invited
• [Presentation] A personal reflection on the Replicon Theory: from R1 plasmid to replication timing regulation in human cells” Symposium “Half a Century With Replicon Theory for Genome Stability and Instability (2013)
  • Author(s): Hisao Masai
  • Organizer: The 50th anniversary of the replicon theory
  • Place of Presentation: Pasteur Institute, Paris, France
  • Invited
• [Presentation] Regulation of replication program in fission yeast and human cells (2012)
  • Author(s): Hisao Masai
  • Organizer: 1st Igakuken Symposium on"Regulation of Chromosome Cycle"
  • Place of Presentation: Igakuken, Tokyo
  • Year and Date: 2012-11-29
  • Invited
• [Presentation] Regulation of replication fork in maintenance of genomic integrity (2012)
  • Author(s): Hisao Masai
  • Organizer: (2012 Arthur Kornberg Memorial Award Lecture) 15th A-IMBN Annual Conference
  • Place of Presentation: Shanghai
  • Year and Date: 2012-10-23
  • Invited
• [Presentation] Regulation of replication program in fission yeast and human cell (2012)
  • Author(s): Hisao Masai
  • Organizer: Department of Biological Science
  • Place of Presentation: Florida State University
  • Year and Date: 2012-09-16
  • Invited
• [Presentation] Cell cycle regulation of mouse embryonic stem cells : E2F- and Emi1-mediated positive feedback regulation (2012)
  • Author(s): Hisao Masai, Hiroko Fujii, Naoko Kakusho, Sayuri Ito, Satoshi Yamazaki, and Naoko Yoshizawa
  • Organizer: 4th Southeast Stem Cell Consortium Workshop at Florida State University
  • Place of Presentation: Tallahassee, Florida
  • Invited
• [Presentation] Regulation of replication program in fission yeast and human cells (2012)
  • Author(s): Hisao Masai, Satoshi Yamazaki, Yutaka Kanoh, Motoshi Hayano, Seiji Matsumoto, Naoko Yoshizawa, Rino Fukatsu, Naoko Kakusho, and Michie Shimmoto
  • Organizer: 8th 3R meeting
  • Place of Presentation: Awajishima
  • Invited
• [Presentation] 解体再構成的アプローチから合成生物学・生命現象シミュレーションへ : DNA複製をモデルとして (2012)
  • Author(s): 正井 久雄
  • Organizer: 第85回日本生化学会大会 シンポジウム
  • Place of Presentation: 福岡
  • Invited
• [Presentation] 解体再構成的アプローチから合成生物学•生命現象シミュレーションへ：DNA複製をモデルとして (2012)
  • Author(s): 正井久雄
  • Organizer: 第85回日本生化学会大会 シンポジウム
  • Place of Presentation: 福岡
  • Invited
• [Presentation] Regulation of replication fork in maintenance of genomic integrity (2012)
  • Author(s): Hisao Masai
  • Organizer: 15th A-IMBN Annual Conference
  • Place of Presentation: Shanghai
  • Invited
• [Presentation] Cell cycle regulation of mouse embryonic stem cells: E2F- and Emi1-mediated positive feedback regulation. (2012)
  • Author(s): Shanghai
  • Organizer: 4th Southeast Stem Cell Consortium Workshop at Florida State University
  • Place of Presentation: Tallahassee, Florida
  • Invited
• [Presentation] Regulation of the replication program in fission yeast and human cells (2011)
  • Author(s): Hisao Masai
  • Organizer: Seminar
  • Place of Presentation: Hong Kong University of Science and Technology
  • Year and Date: 2011-11-25
  • Invited
• [Presentation] Regulation of replication program in fission yeast and human cells (2011)
  • Author(s): Hisao Masai, Satoshi Yamazaki, Yutaka Kanoh, Motoshi Hayano, Seiji Matsumoto, Naoko Kakusho
  • Organizer: Cold Spring Harbor Laboratory Meeting Eukaryotic DNA Replication & Genome Maintenance
  • Place of Presentation: New York, USA
  • Year and Date: 2011-09-06
• [Presentation] DNA 複製制御因子を標的とした制がん戦略 : Cdc7 キナーゼ阻害によるがん細胞死誘導 (2011)
  • Author(s): 正井久雄
  • Organizer: 第9回日本臨床腫瘍学会/日本癌学会/分子標的治療学会 合同シンポジウム"基礎から学ぶ次世代の分子標的治療薬と基盤技術"
  • Place of Presentation: 東京
  • Year and Date: 2011-07-21
  • Invited
• [Presentation] Regulation of replication program in fission yeast (2011)
  • Author(s): Hisao Masai, Seiji Matsumoto, Motoshi Hayano, Yutaka Kanoh, Michie Shimmoto, Naoko Kakusho, and Satomi Kudo
  • Organizer: The 6th International Fission Yeast Meeting
  • Place of Presentation: Boston, USA
  • Invited
• [Presentation] Rif1 is a global regulator of site selection and timing of replication origin firing (2011)
  • Author(s): Motoshi Hayano, Yutaka Kanoh, Seiji Matsumoto and Hisao Masai
  • Organizer: The 6th International Fission Yeast Meeting
  • Place of Presentation: Boston, USA
• [Presentation] "Regulation of replication program in fission yeast and human cells." (2011)
  • Author(s): Hisao Masai, Satoshi Yamazaki, Yutaka Kanoh, Motoshi Hayano, Seiji Matsumoto, Masako Oda, Naoko Kakusho, Rino Fukatsu, Michie Shimmoto and Naoko Yoshizawa
  • Organizer: 日本分子生物学会総会 シンポジウム(招待講演)
  • Place of Presentation: 横浜
• [Presentation] DNA複製制御因子を標的とした制がん戦略：Cdc7キナーゼ阻害によるがん細胞死誘導 (2011)
  • Author(s): 正井久雄
  • Organizer: 第9回 日本臨床腫瘍学会/日本癌学会/分子標的治療学会 合同シンポジウム "基礎から学ぶ次世代の分子標的治療薬と基盤技術"(招待講演)
  • Place of Presentation: 東京
• [Book] 細胞工学 (2012)
  • Author(s): 早野元詞、加納豊、松本清治、正井久雄
  • Total Pages: 490
  • Publisher: 秀潤社
• [Remarks]
  • URL: http://www.igakuken.or.jp/genome/