ヒト細胞遺伝子のノックアウトの高効率化のための萌芽的研究

• Project/Area Number: 18657053
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Tohoku University
• Principal Investigator: 関 政幸 Tohoku University, 大学院・薬学研究科, 准教授 (70202140)
• Project Period (FY): 2006 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: 相同組換え / Ino80 / Arp4 / Arp6 / Swr1 / Rad52 / NHEJ / CAF-1 / ChIP / 出芽酵母 / 標的組換え / ヒストン

Research Abstract

1)出芽酵母Arp6は組換えの抑制に関わる;Swr1複合体のサブユニットarp6変異株で組換えが亢進することを論文発表した(Kawashima et al.,2007)。
2)出芽酵母を用いた、相同組換えのクロマチンレベルでの抑制機構の解明;sgs1変異株において相同組換えの亢進(swr1株よりも亢進の程度は低い)が観察される。ここで、swr1-sgs1二重変異株を作製して、今までに報告のない高い頻度の組換えが起るかどうかを調べたが、予想に反してそうはならなかった。一方、複製にカップルしてヌクレオソームの形成に関わるCAF-1とSgs1の同時欠損において、相乗的な姉妹染色分体間の組換えの亢進を見いだした。
3)ヒトNalm6細胞を用いた、高効率の遺伝子破壊法の確立;ヒトNalm6細胞はB細胞由来の細胞株で、比較的標的遺伝子の破壊の効率が良い細胞として知られ、既に幾つかの遺伝子破壊株の作製とその解析が報告されている。そこでSWR1とBLMの両遺伝子の発現を同時に抑制させるsiRNAをNalm6細胞に導入し、その条件下で標的遺伝子の破壊効率の測定を行う予定だったが、出芽酵母swr1-sgs1株でhyper-recombinationの表現型が観察されなかったため中断した。その代わりニワトリDT40細胞でCAF1およびBLMのノックアウト細胞が確立しているのを利用し、DT40 BLM-CAF1二重遺伝子破壊株を作製している。この株を用いて、標的遺伝子破壊の上昇を確かめ、その後Nalm6系への応用を想定して実験を進めている。

Research Products

• [Journal Article] Vertebrate WRNIPI and BLM are required for efficient maintenance of genome stability (2008)
  • Author(s): Hayashi T, Seki M, Inoue E, Yoshimura A, Kusa Y, Tada S, Enomoto T
  • Journal Title: Genes Genet. Syst. 83 Pages: 95-100
  • Peer Reviewed
• [Journal Article] Analyses of functional interaction between RECQLI, RECQL5, and BLM which physically interact with DNA tonoisomerase IIIa (2008)
  • Author(s): Otsuki M, Seki M, Inoue E, Abe T, Narita Y, Yoshimura A, Tada S, Ishii Y, Enomoto T
  • Journal Title: Bioehim. Biophys. Acta. 1782 Pages: 75-81
  • Peer Reviewed
• [Journal Article] Functional interactions between BLM and XRCC3 in the cell (2007)
  • Author(s): Otsuki M, Seki M, Inoue E, Yoshimura A, Kato G, Yanumouchi S, Kawabe Y, Tada S, Shinohara A, Komura J, Ono T, Takeda S, Ishii Y, Enomoto T.
  • Journal Title: J. Cell Biol. 179 Pages: 53-63
  • Peer Reviewed
• [Journal Article] Ctf18 is required for homologous recombination-mediated double-strand break repair (2007)
  • Author(s): Ogiwara. H., Ohuchi, T, , Ui, A., Tada, S., Enomoto, T., Seki, M.
  • Journal Title: Nucleic Acids Res. 35 Pages: 4989-5000
  • Peer Reviewed
• [Journal Article] Rmil, a member of the Sgs1-Top3 complex in budding yeast, contributesto sister chromatid cohesion (2007)
  • Author(s): Lai, M.S., Seki, M., Ui, A., Enomoto, T.
  • Journal Title: EMBO Rep. 8 Pages: 685-690
  • Peer Reviewed
• [Journal Article] Activation of a novel pathway involving Mms1 and Rad59 in sgs1 cells (2007)
  • Author(s): Ui A, Seki M, Ogiwara H, Lai MS, Yamamoto K, Tada S, Enomoto T.
  • Journal Title: Biochem. Biophys. Res. Commun. 356 Pages: 1031-1037
  • Peer Reviewed
• [Journal Article] The INO80 complex is required for damage-induced recombination (2007)
  • Author(s): Kawashima S, Ogiwara H, Tada S, Harata M, Wintersberger U, Enomoto T, Seki M.
  • Journal Title: Biochem. Biophys. Res. Commun. 355 Pages: 835-841
  • Peer Reviewed
• [Journal Article] Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopusegg extracts (2007)
  • Author(s): Kumata Y, Tada S, Yamanada Y, Tsuyama T, Kobayashi T, Dong YP, Ikegami K, Murofushi H, Seki M, Enomoto T.
  • Journal Title: Biochim. Biophys. Acta. 1773 Pages: 556-564
  • Peer Reviewed
• [Journal Article] WRN counteracts the NHEJ pathway upon camptothecin exposure (2007)
  • Author(s): Otsuki M, Seki M, Kawabe Y, Inoue E, Dong YP, Abe T, Kato G, Yoshimura A, Tada S, Enomoto T.
  • Journal Title: Biochem. Biophys. Res. Commun. 355 Pages: 477-482
  • Peer Reviewed
• [Journal Article] WRN functions in a RAD18-dependent damage avoidance pathway (2007)
  • Author(s): Dong YP, Seki M, Yoshimura A, Inoue E, Furukawa S, Tada S, Enomoto T
  • Journal Title: Biol. Pharm. Bull. 30 Pages: 1080-1083
  • NAID: 110006278395
  • Peer Reviewed
• [Journal Article] Rad50 is involved in MMS-induced recombination between homologous chromosomes in mitoticcells (2007)
  • Author(s): Tomizawa Y, Ui A, Onoda F, Ogiwara H, Tada S, Enomoto T, Seki M.
  • Journal Title: Genes Genet. Syst. 82 Pages: 157-160
  • Peer Reviewed
• [Journal Article] The INO80 chromatin remodeling complex functions in sister chromatid cohesion (2007)
  • Author(s): Ogiwara H, Enomoto T, Seki M.
  • Journal Title: Cell Cycle 6 Pages: 1090-1095
  • Peer Reviewed
• [Journal Article] Actin-related protein Arp4 functions in kinetochore assembly (2007)
  • Author(s): Ogiwara H, Ui A, Kawashima S, Kugou K, Onoda F, Iwahashi H, Harata M, Ohta K, Enomoto T, Seki M.
  • Journal Title: Nucleic Acids Res. 35 Pages: 3109-3117
  • Peer Reviewed
• [Journal Article] Role of Elg1 protein in double strand break repair (2007)
  • Author(s): Ogiwara, H., Ui, A., Enomoto, T., Seki, M.
  • Journal Title: Nucleic Acids Res. 35 Pages: 353-362
• [Journal Article] The INO80 complex is required for damage-induced recombination (2007)
  • Author(s): Kawashima, S., Ogiwara, H., Tada, S., Harata, M., Wintersberger, U., Enomoto T., Seki, M.
  • Journal Title: Biochem. Biophys. Res. Commun. 355 Pages: 835-841
• [Journal Article] Bloom helicase and DNA topoisomerase IIIα are involved in the dissolution of sister chromatids (2006)
  • Author(s): Seki, M., Nakagawa, T., Seki, T., Kato, G., Tada, T., Takahashi, Y., Yoshimura, A., Kobayashi, T., Aoki, A., Otsuki, M., Habermann, F.A., Tanabe, H., Ishii, Y., Enomoto, T.
  • Journal Title: Mol. Cell. Biol. 26 Pages: 6299-6307
• [Journal Article] Ubc9 and Mms21 mediated sumoylation counteracts recombinogenic events at damaged replication forks (2006)
  • Author(s): Branzei, D., Sollier, J., Liberi, G., Zhao, X., Maeda, D., Seki, M., Enomoto, T., Ohta, K., Foiani, M.
  • Journal Title: Cell 127 Pages: 509-522
• [Journal Article] BLM is an early responder to DNA double-strand breaks (2006)
  • Author(s): Karmakar, P., Seki, M., Kanamori, M., Hashiguchi, K., Ohtsuki, M., Murata, E., Inoue, E., Tada, S., Lan, L., Yasui, A., Enomoto, T.
  • Journal Title: Biochem. Biophys. Res. Commun. 348 Pages: 62-69
• [Journal Article] Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature /cold sensitive alleles of the DNA polymerase δ subunit, Pol31 (2006)
  • Author(s): Davoodi Vijeh Motlagh, N., Seki, M., Branzei, D., Enomoto, T.
  • Journal Title: DNA Repair 5 Pages: 1459-1474
• [Presentation] Requirement of specific amino acids in Rad52 for sumoylation and recombination (2007)
  • Author(s): 関 政幸
  • Organizer: 第三回Japan-US DNA Repair Meeting
  • Place of Presentation: 仙台市/秋保/ホテルクレセント
  • Year and Date: 2007-05-09
• [Book] Reviews and Protocols in DT40 Research (2006)
  • Author(s): 関政幸/多田周右/榎本武美
  • Total Pages: 24
  • Publisher: Springer社