停止複製フォーク結合タンパク質PriAを用いた染色体脆弱部位の探索

• Project/Area Number: 21657036
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Functional biochemistry
• Research Institution: Tokyo Metropolitan Organization for Medical Research
• Principal Investigator: 正井 久雄 (財)東京都医学研究機構, 東京都臨床医学総合研究所, 参事研究員 (40229349)
• Co-Investigator(Kenkyū-buntansha): 田中 卓 (財)東京都医学研究機構, 東京都臨床医学総合研究所, 研究員 (80425686) ; 加納 豊 (財)東京都医学研究機構, 東京都臨床医学総合研究所, 研究員 (90450593)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥3,100,000 (Direct Cost: ¥3,100,000); Fiscal Year 2010: ¥1,500,000 (Direct Cost: ¥1,500,000); Fiscal Year 2009: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: PriAタンパク / 複製フォーク / 染色体脆弱部位 / 染色体免疫沈降 / aphidicolin / ChIP-chip解析 / DNA複製停止 / DNAヘリカーゼ

Research Abstract

本研究では、大腸菌PriAタンパク質の「停止した複製フォークに選択的に強く結合する」という生化学的性質をtoolとして、複製が停止しやすいあるいはslow downする染色体脆弱部位を染色体上の複製が停止し易い領域あるいは、停止した際に適切に回復さない領域として網羅的に探索することを計画した。
動物細胞(がん細胞あるいは正常細胞)でPriAタンパクを発現し、正常に複製している状態あるいは低濃度のaphidicolinあるいはHU(ヒドロキシ尿素:複製阻害剤)で複製進行を阻害した場合にPriAタンパク質が結合するDNAを染色体免疫沈降法で濃縮し、同定する。期間内に、このシステムを構築し、複製停止部位をゲノムワイドで同定する。種々のヒト細胞でtag付きPriAをectopicに発現する細胞株を樹立し、tagに対する抗体を用いた染色体免疫沈降法により、沈降されるDNAを濃縮、増幅し、ゲノムタイリングアレイへのhybridizationあるいは網羅的塩基配列決定により、複製フォークの停止し易い領域をゲノムワイドで同定する計画である。本年度の成果は以下の通りである。
1 作製したPriAに対する特異抗体および付加したtagに対する抗体により、様々な細胞(ヒトがん細胞および正常細胞)に発現したPriAを免疫沈降できることを確認した。また、クロスリンクによる染色体免疫沈降条件下でも解析可能な効率で免疫沈降できることを確認した。マイクロアレイ解析に持ち込める染色体を沈降させるための条件検討を重ね、最適条件を決定した。
2 まず大腸菌をもちいて、染色体免疫沈降を行い、マイクロアレイによる検出を試みた。コントロールとして、PriA欠損株のほか、複製起点や複製中のフォークに存在するDnaBなど、複製装置を構成するタンパク質を利用した。これらの大腸菌のゲノム上に検出される各ピークの解析において、既存のソフトウェアでは結合イベントの妥当性を正確に見積もれなかったため、専用のアルゴリズムを作製し、現在検証中である。

Research Products

• [Journal Article] Pre-firing binding of Mrcl 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., Masai, H.
  • Journal Title: Mol.Cell.Biol. Volume: 31 In press
  • Peer Reviewed
• [Journal Article] Efficient expression and purification of human replication fork-stabilizing factor, Claspin, from mammalian cells : DNA binding activity and novel protein interactions. (2011)
  • Author(s): Uno, S, Masai, H.
  • Journal Title: Genes to Cells Volume: 16 In press
  • Peer Reviewed
• [Journal Article] Molecular mechanism of activation of human Cdc7 kinase : Bipartite inter action with Dbf4/ASK stimulates ATP binding and substrate recognition. (2011)
  • Author(s): Ryo Kitamura, Rino Fukatsu, Naoko Kakusho, Yong-Soon Cho, Chika Taniyama, Satoshi Yamazaki, Gaik-theng Toh, Kazuo Yanagi, Naoko Arai, Ho-Jin Chang, Hisao Masai
  • Journal Title: J.Biol.Chem. Volume: 286 In press
  • Peer Reviewed
• [Journal Article] RecQL4 : a helicase linking formation and maintenance of a replication fork. (2011)
  • Author(s): Masai, H.
  • Journal Title: J.Biochem. Volume: 142 In press
  • NAID: 10030585512
  • Peer Reviewed
• [Journal Article] Cdc7L1. (2011)
  • Author(s): Toh G-T., Masai, H.
  • Journal Title: UCSD-Nature Molecule Pages Volume: In press
  • Peer Reviewed
• [Journal Article] Fission yeast Swil-Swi3 complex facilitates DNA binding of Mrc1. (2010)
  • Author(s): Tanaka, T., Yokoyama, M., Matsumoto, S., Fukatsu, R., You, Z., Masai, H.
  • Journal Title: J.Biol.Chem. Volume: 285 Pages: 39609-39622
  • Peer Reviewed
• [Journal Article] Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex. (2010)
  • Author(s): Kundu, L.R., Kumata, Y., Kakusho, N., Watanabe, S., Furukohri, A., Waga, S., Sekia, M., Masai, H., Enomoto, T., Tada, S.
  • Journal Title: Nucleic Acid Res. Volume: 38 Pages: 5409-5418
  • Peer Reviewed
• [Journal Article] Casein kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1. (2010)
  • Author(s): Takeishi, Y., Ohashi, E., Ogawa, K, Masai, H., Obuse, C., Tsurimoto, T.
  • Journal Title: Genes Cells Volume: 15 Pages: 761-771
  • NAID: 120005227474
  • Peer Reviewed
• [Journal Article] DDK phosphorylates checkpoint clamp Rad9 and promotes its release from damaged Chromatin. (2010)
  • Author(s): Furuya, K., Miyabe, I., Tsutsui, Y., Paderi, F., Kakusho, N., Masai, H., Niki, H., Carr, A.M.(20
  • Journal Title: Mol.Cell Volume: 40 Pages: 606-618
  • Peer Reviewed
• [Journal Article] Cdc7-Mediated Rad18 Phosphorylation Directs the Accumulation of DNA Polymerase η at Sites of Stalled Replication. (2010)
  • Author(s): Day, T.A., Palle, K., Barkley, L.R., Kakusho, N., Zou, Y., Tateishi, S., Verreault, A., Masai, H., Vaziri, C.
  • Journal Title: J.Cell Biol. Volume: 191 Pages: 953-966
  • Peer Reviewed
• [Journal Article] Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast. (2010)
  • Author(s): Matsumoto, S., Shimmoto, M., Kakusho, N., Yokoyama, M., Russell, P., Masai, H.
  • Journal Title: Cell Cycle Volume: 9 Pages: 4627-4637
  • Peer Reviewed
• [Journal Article] FANCs regulate firing of DNA replication origins. (2010)
  • Author(s): Masai, H.
  • Journal Title: Cell Cycle Volume: 9 Pages: 2494-2494
  • Peer Reviewed
• [Journal Article] Integrating DNA replication with Trans-Lesion Synthesis via Cdc7. (2010)
  • Author(s): Vaziri, C., Masai H.
  • Journal Title: Cell Cycle Volume: 9 Pages: 4818-4823
  • Peer Reviewed
• [Journal Article] ASK. (2010)
  • Author(s): Toh G-T., Masai, H.
  • Journal Title: UCSD-Nature Molecule Pages(Review) Volume: (Published online: 25 Feb 2009)
  • Peer Reviewed
• [Journal Article] Eukaryotic DNA replication ; where, when and how? (2010)
  • Author(s): Masai, H., Matsumoto, S., You, Z., Sugata-Yoshizawa, N., Oda, M.
  • Journal Title: Annual.Rev.Biochem. Volume: 79 Pages: 89-130
  • Peer Reviewed
• [Journal Article] Stalled replication forks : Making ends meet for recognition and stabilization. (2010)
  • Author(s): Masai, H., Tanaka, T., Kohda, D.
  • Journal Title: BioEssays Volume: 32 Pages: 687-697
  • Peer Reviewed
• [Journal Article] Bacterial primosome. (2010)
  • Author(s): Tanaka, T., Masai, H.
  • Journal Title: Encyclopedia of Life Sciences
  • Peer Reviewed
• [Journal Article] Bacterial replication fork. (2010)
  • Author(s): Tanaka, T., Masai, H.
  • Journal Title: Encyclopedia of Life Sciences
  • Peer Reviewed
• [Journal Article] Eukaryotic DNA replication ; where, when and how? (2010)
  • Author(s): Masai, H., Matsumoto, S., You, Z., Sugata-Yoshizawa, N., Oda, M.
  • Journal Title: Annual.Rev.Biochem. (In press)
  • Peer Reviewed
• [Journal Article] Selection of replication origins in fission yeast (2010)
  • Author(s): Matsumoto, S., Hayano, M., Kanoh, Y., Masai, H.
  • Journal Title: Chromosoma (To be published)
  • Peer Reviewed
• [Journal Article] Cdc7L1 (2010)
  • Author(s): Toh G-T., Masai, H.
  • Journal Title: UCSD-Nature Molecule Pages (In press)
  • Peer Reviewed
• [Journal Article] Stalled replication forks : Making ends meet for recognition and stabilization (2010)
  • Author(s): Masai, H., Tanaka, T., Kohda, D.
  • Journal Title: BioEssays (In press)
  • Peer Reviewed
• [Journal Article] Bacterial primosome (2010)
  • Author(s): Tanaka, T., Masai, H.
  • Journal Title: Encyclopedia of Life Sciences (in press)
  • Peer Reviewed
• [Journal Article] Bacterial replication fork (2010)
  • Author(s): Tanaka, T., Masai, H.
  • Journal Title: Encyclopedia of Life Sciences (in press)
  • Peer Reviewed
• [Journal Article] Interactions between Swil-Swi3, Mrc1 and S phase kinase, Hsk1 may regulate cellular responses to stalled replication forks in fission yeast (2009)
  • Author(s): Shimmoto, S., Matsumoto, S., Hayano, M., Yokoyama, M., Noguchi, E., Russell, P., Masai, H.
  • Journal Title: Genes to Cells 14 Pages: 669-682
  • Peer Reviewed
• [Journal Article] Replisome progression complex links DNA replication to sister chromatid cohesion in Xenopus egg extracts (2009)
  • Author(s): Tanaka, H., Kubota, Y., Tsujimura, T., Kumano, M., Masai, H., Takisawa, H.
  • Journal Title: Genes to Cells 14 Pages: 949-963
  • Peer Reviewed
• [Journal Article] Roles of human AND-1 in chromosome transactions in S phase (2009)
  • Author(s): Yoshizawa-Sugata, N., Masai, H.
  • Journal Title: J.Biol.Chem. 284 Pages: 20718-20728
  • Peer Reviewed
• [Journal Article] Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex (2009)
  • Author(s): Kundu, L.R., Kumata, Y, Kakusho, N., Watanabe, S., Furukohri, A., Waga, S., Sekia, M., Masai, H., Enomoto, T., Tada, S.
  • Journal Title: Nucleic Acid Res. (In press)
  • Peer Reviewed
• [Journal Article] Casein Kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1 (2009)
  • Author(s): Takeishi, Y., Ohashi, E., Ogawa, K., Masai, H., Obuse, C., Tsurimoto, T.
  • Journal Title: Genes to Cells (In press)
  • NAID: 120005227474
  • Peer Reviewed
• [Journal Article] Drug Design with Cdc7 kinase, a potential novel cancer therapy target (2009)
  • Author(s): Sawa, M., Masai, H.
  • Journal Title: Drug Design, Development and Therapy 2 Pages: 255-264
  • Peer Reviewed
• [Journal Article] ASK (2009)
  • Author(s): Toh, G.K., Masai, H.
  • Journal Title: UCSD-Nature Molecule Pages (On line)
  • Peer Reviewed
• [Presentation] Toward understanding the regulation of chromosome cycle by Cdc7 kinase. (2010)
  • Author(s): Masai, H.(Organizer and speaker)
  • Organizer: The final symposium on Chromosome Cycle
  • Place of Presentation: Hongo, Tokyo, Japan(招待講演)
  • Year and Date: 2010-10-01
• [Presentation] Regulation of replication program and maintenance of genetic integrity. (2010)
  • Author(s): Masai, H., Yamazaki, S., Hayano, M., Kanoh, Y., Matsumoto, S., Kakusho, N., Ishii, A., Shimmoto, M., Oda, M., Yoshizawa, N., Ito, S., Kudo, S.(Organizer and speaker)
  • Organizer: BMB2010 Symposium
  • Place of Presentation: Kobe, Japan(招待講演)
• [Presentation] Cell Cycle and Mitosis. (2010)
  • Author(s): Masai, H.
  • Organizer: Karolinska Institute RIKEN Joint Course 'Functional architecture of the cell nucleus
  • Place of Presentation: RIKEN Yokohama(招待講演)(Invited lecture)
• [Presentation] Cell cycle regulation of embryonic stem cells regulating the maintenance of pluripotency and differentiation. (2010)
  • Author(s): Masai, H., Fujii, H., Kanoh, Y., Kakusho, N., Ito, S., Sawano, A., Miyawaki, A.
  • Organizer: The 53^<rd> annual meeting of the Japan Radiation Society.(日本放射線影響学会)
  • Place of Presentation: Kyoto, Japan(招待講演)
• [Presentation] 未分化胚性幹細胞の自己増幅と分化誘導を制御する細胞周期制御機構 (2010)
  • Author(s): 正井久雄、藤井裕子、加納豊、伊藤さゆり、沢野朝子、宮脇敦史
  • Organizer: 第9回日本再生医療学会総会
  • Place of Presentation: 広島
• [Presentation] 複製開始部位の選択と活性化タイミングの遺伝的制御と可塑性 (2009)
  • Author(s): 正井久雄
  • Organizer: 日本遺伝学会第81回大会シンポジウム「日本のDNA複製研究の過去、現在、未来」
  • Place of Presentation: 信州大学理学部(長野県松本市)
• [Presentation] Actions of Mcm helicase and fork stabilizing factors at replication forks (2009)
  • Author(s): Masai, H., You, Z., Tanaka, T., Yokoyama, M.
  • Organizer: 第82回日本生化学会大会
  • Place of Presentation: 神戸
• [Presentation] Cellular responses to stalled replication forks : roles of Cdc7 kinase and fork stabilization factors (2009)
  • Author(s): Masai, H.
  • Organizer: 25th RBC International Symposium "Genomic Instability : mechanisms and diseases"
  • Place of Presentation: Kyoto
• [Presentation] EBNA1-dependentassociation of mammalian pre-RC components with oriP of Epstein-Barr Virus in vitro (2009)
  • Author(s): 森山賢治, 高井裕子, 正井久雄
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 横浜
• [Presentation] Factors Regulating the Origin Selection in Eukaryotic DNA Replication (2009)
  • Author(s): Masai, H., Matsumoto, S., Hayano, M., Kanoh, Y., Yamazaki, S., Shimmoto, M., Kakusho, N., Kudo, S.
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 横浜
• [Book] 細胞周期の時空間的解析(細胞周期フロンティア)(佐方功幸、稲垣昌樹、岸本健雄編集) (2010)
  • Author(s): 阪上-沢野朝子、正井久雄、宮脇敦史
  • Total Pages: 7
  • Publisher: 共立出版
• [Book] なぜ親子は似るの?(なぜなぜ生物学)(MBSJ日本分子生物学会編) (2010)
  • Author(s): 正井久雄
  • Total Pages: 15
  • Publisher: 東京化学同人
• [Book] 生物学辞典 (分担) (2010)
  • Author(s): 正井久雄
  • Publisher: 東京化学同人
• [Book] 序蛋白質核酸酵素増刊号「染色体サイクル」 (2009)
  • Author(s): 正井久雄
  • Total Pages: 1
  • Publisher: 共立出版
• [Book] 序論 染色体サイクルの基礎. 蛋白質核酸酵素 増刊号「染色体サイクル」 (2009)
  • Author(s): 正井久雄
  • Total Pages: 6
  • Publisher: 共立出版
• [Book] 染色体サイクルの連係的制御機構 概論 蛋白質核酸酵素 増刊号「染色体サイクル」 (2009)
  • Author(s): 正井久雄
  • Total Pages: 3
  • Publisher: 共立出版
• [Book] Cdc7キナーゼによる染色体サイクル制御. 蛋白質核酸酵素 増刊号「染色体サイクル」 (2009)
  • Author(s): 正井久雄
  • Total Pages: 7
  • Publisher: 共立出版
• [Book] S期開始, 進行の制御と疾患. 炎症と免疫 (2009)
  • Author(s): 正井久雄
  • Total Pages: 8
  • Publisher: 先端医学社
• [Book] 細胞周期を時空間的に可視化する技術. 細胞工学、「細胞周期研究の新たなステージ」 (2009)
  • Author(s): 阪上-沢野朝子、正井久雄、宮脇敦史
  • Total Pages: 8
  • Publisher: 秀潤社
• [Book] 細胞周期を時空間的に可視化する技術. 最新医学 増刊号 「幹細胞研究の最近の進歩」 (2009)
  • Author(s): 阪上-沢里朝子、正井久雄、宮脇敦史
  • Total Pages: 4
  • Publisher: 最新医学社
• [Remarks]
  • URL: http://www.rinshoken.or.jp/CB/index-jp.htm