染色体の核内高次構築を支配する非コード領域とそれを制御するタンパク質の解析

• Project Area: Functions of non-coding DNA region for genome integrity
• Project/Area Number: 24114520
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: 正井 久雄 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)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥12,220,000 (Direct Cost: ¥9,400,000、Indirect Cost: ¥2,820,000); Fiscal Year 2013: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000); Fiscal Year 2012: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
• Keywords: 染色体 / ゲノム / 核 / DNA複製 / 転写 / グアニン４重鎖 / クロマチンループ / 非コード領域 / 複製起点 / 複製タイミング / Rif1タンパク質 / Cdc7キナーゼ / pre-RC / 複製ストレス / チェックポイント応答

Outline of Annual Research Achievements

組換えRif1タンパク質の発現と精製を行い、Rif1タンパク質によるクロマチン制御をタンパク質-核酸相互作用レベルで解析し、Rif1タンパク質によるクロマチン制御をタンパク質-核酸相互作用レベルで調べる、という目的を達成するために、分裂酵母Rif1タンパク質を動物細胞で発現し、affinity columnなどにより精製した。精製Rif1タンパク質を用いて、これがDNAに結合すること、特にグアニン４重鎖構造を有するDNAに特異的に結合することを発見した。
この発見は核内染色体構築において、遺伝子間領域に存在するグアニン４重鎖構造の形成と、Rif1のそれらへの結合が決定的な役割を果たすことを示唆する。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Research Products

• [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: 1170 Pages: 279-293
  • DOI: 10.1007/978-1-4939-0888-2_13
  • ISBN: 9781493908875, 9781493908882
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] ATM in prevention of genomic instability. (2014)
  • Author(s): Hisao Masai
  • Journal Title: Cell Cycle Volume: 13 Issue: 6 Pages: 882-883
  • DOI: 10.4161/cc.28216
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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.
  • Journal Title: Seminars in Cell & Developmental Biology, Volume: 30 Pages: 110-120
  • DOI: 10.1016/j.semcdb.2014.04.014
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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 Issue: 22 Pages: 2459-72
  • DOI: 10.1101/gad.224568.113
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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 Issue: 18 Pages: 12742-12752
  • DOI: 10.1074/jbc.m112.398073
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation. (2013)
  • Author(s): Jeffery, D.C., Wyse, B.A., Rehman, M.A., Brown, G.W., You, Z., Oshidari, R., Masai, H. and Yankulov, K.Y.
  • Journal Title: Nucleic Acids Res. Volume: 41 Issue: 18 Pages: 8475-8488
  • DOI: 10.1093/nar/gkt623
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair (2013)
  • Author(s): Tanikawa, M., Wada-Hiraike, O., Yoshizawa-Sugata, N., Shirane A, Hirano M, Hiraike H, Miyamoto, Y., Sone, K., Ikeda, Y., Kashiyama, T., Oda K, Kawana K, Katakura Y, Yano T, Masai, H., Roy AL, Osuga, Y. and Fujii, T.
  • Journal Title: Br. J. Cancer. Volume: 109 Issue: 12 Pages: 3042
  • DOI: 10.1038/bjc.2013.532
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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 Issue: 8 Pages: 449-460
  • DOI: 10.1016/j.tig.2013.05.001
  • Peer Reviewed / Open Access
• [Journal Article] A personal reflection on the Replicon Theory: from R1 plasmid to replication timing regulation in human cells (2013)
  • Author(s): Masai, H.
  • Journal Title: J. Mol. Biol. Volume: 425 Issue: 23 Pages: 4663-4672
  • DOI: 10.1016/j.jmb.2013.03.039
  • 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 Issue: 6 Pages: 1712-1719
  • DOI: 10.1042/bst20130217
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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
  • Peer Reviewed
• [Journal Article] Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob. (2012)
  • Author(s): Suzuki T
  • Journal Title: J Biol Chem Volume: Nov 23;287(48) Issue: 48 Pages: 40256-40265
  • DOI: 10.1074/jbc.m112.353805
  • Peer Reviewed
• [Journal Article] A central coupler for recombination initiation linking chromosome architecture to S phase checkpoint. (2012)
  • Author(s): 三好知一郎
  • Journal Title: Molecular Cell Volume: 14 Issue: 5 Pages: 722-733
  • DOI: 10.1016/j.molcel.2012.06.023
  • Peer Reviewed
• [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.
  • Journal Title: PLoS One Volume: 7 Issue: 8 Pages: 214-221
  • DOI: 10.1371/journal.pone.0042375
  • Peer Reviewed
• [Journal Article] Purification of replication factors using insect and mammalian cell expression systems. (2012)
  • Author(s): Uno, S., You, Z., and Masai, H.
  • Journal Title: Methods Volume: 57 Issue: 2 Pages: 214-221
  • DOI: 10.1016/j.ymeth.2012.06.016
  • Peer Reviewed
• [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
  • Journal Title: Genes & Dev Volume: 25 Issue: 2 Pages: 137-150
  • DOI: 10.1101/gad.178491.111
  • Peer Reviewed
• [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 Issue: 18 Pages: 3167-3177
  • DOI: 10.1038/emboj.2012.180
  • Peer Reviewed
• [Journal Article] c-Jun N-terminal Kinase (JNK)-Mediated Rad18 Phosphorylation Facilitates Polη Recruitment to Stalled Replication Forks. (2012)
  • Author(s): Barkley, L.R., Palle, K., Durando, M., Day, T.A., Gurkar, A., Kakusho, N., Li, J., Masai, H., Vaziri, C.
  • Journal Title: Mol. Biol. Cell. Volume: 23 Issue: 10 Pages: 1943-1954
  • DOI: 10.1091/mbc.e11-10-0829
  • Peer Reviewed
• [Journal Article] Epstein-Barr nuclear antigen 1 (EBNA1)-dependent recruitment of origin recognition complex(Orc) on oriP of Epstein-Barr virus with purified proteins:stimulation by Cdc6 through its direct interaction with EBNA1. (2012)
  • Author(s): Moriyama K
  • Journal Title: The Journal of biological chemistry Volume: 287 Issue: 28 Pages: 23977-23994
  • DOI: 10.1074/jbc.m112.368456
  • Peer Reviewed
• [Journal Article] Mechanism of cancer cell death induced by depletion of an essential replication regulator. (2012)
  • Author(s): Ito, S., Ishii, A., Kakusho, N., Taniyama, C., Yamazaki, S., Sakaue-Sawano, A., Miyawaki, A., and Masai, H.
  • Journal Title: PLoS One Volume: 7 Issue: 5 Pages: e36372-e36372
  • DOI: 10.1371/journal.pone.0036372
  • Peer Reviewed
• [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)
  • Author(s): 早野元詞、加納豊、松本清治、正井久雄
  • Journal Title: 細胞工学 Volume: 31 Pages: 460-462
• [Journal Article] Rif1タンパク質はヒトゲノム複製タイミングドメインを決定する。 (2012)
  • Author(s): 山崎 聡志、正井 久雄
  • Journal Title: 実験医学、Current Topics Volume: 30 Pages: 2974-2978
• [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: 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（静岡）
  • Year and Date: 2014-02-13 – 2014-02-14
  • 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』
  • Place of Presentation: 神戸ポートアイランド（兵庫）
  • Year and Date: 2013-12-03 – 2013-12-06
  • 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: 神戸ポートアイランド（兵庫）
  • Year and Date: 2013-12-03 – 2013-12-06
  • Invited
• [Presentation] Introduction" Symposium (2013)
  • Author(s): Hisao Masai
  • Organizer: 86th Annual Meeting of the Japanese Biochemical Society
  • Place of Presentation: パシフィコ横浜（神奈川）
  • Year and Date: 2013-09-11 – 2013-09-13
  • 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: パシフィコ横浜（神奈川）
  • Year and Date: 2013-09-11 – 2013-09-13
  • 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
  • Year and Date: 2013-06-24 – 2013-06-29
  • Invited
• [Presentation] A personal reflection on the Replicon Theory: from R1 plasmid to replication timing regulation in human cells (2013)
  • Author(s): Hisao Masai
  • Organizer: Half a Century With Replicon Theory for Genome Stability and Instability
  • Place of Presentation: Pasteur Institute, Paris, France
  • Year and Date: 2013-03-25
  • 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
  • Year and Date: 2013-03-21
  • Invited
• [Presentation] 解体再構成的アプローチから合成生物学#8226;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
  • Year and Date: 2012-12-14
  • Invited
• [Presentation] Cell cycle regulation of mouse embryonic stem cells: E2F- and Emi1-mediated positive feedback regulation. (2012)
  • Author(s): Hisao Masai
  • Organizer: 4th Southeast Stem Cell Consortium Workshop at Florida State University
  • Place of Presentation: Tallahassee, Florida
  • Year and Date: 2012-09-28
  • 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