DNA修復機構の、逆遺伝学的手法(ニワトリ細胞株とメダカ)による機能解析

2007 Fiscal Year Annual Research Report

• Project/Area Number: 17013039
• Research Institution: Kyoto University
• Principal Investigator: 武田 俊一 Kyoto University, 医学研究科, 教授 (60188191)
• Co-Investigator(Kenkyū-buntansha): 谷口 善仁 京都大学, 医学研究科, 助教 (00324616)
• Keywords: DNA組み換え / DNA修復 / 変異 / ゲノム不安定性 / シスプラチン / DT40 / メダカ / p53

Research Abstract

#プロテオソーム阻害剤の抗ガン作用機序の解明(Murakawa, et. al., Cancer Res. 2007)
我々は、Mol Cell 2007年に、E2ユビキチンリガーゼ、Ubc13が相同組み換えに関与していることを報告した。Ubc13の作用機序を解明する為に、プロテオソームが相同組み換えに関与するか否かを解析した。その結果、Ubc13と同じステップ(DNA切断端から単鎖を形成するステップ)でプロテオソームが相同組み換えを促進することがわかった。
相同組み換えは、抗がん剤、シスプラチンやカンプトテシン処理によって生じたDNA損傷を修復する。よって、これらの抗がん剤とプロテオソーム阻害剤とを併用すると、抗がん剤の作用を増強できる。
#P53依存性の、精子DNAにおける変異抑制の解明
メダカやゼブラフィッシュでは、ES細胞がない。そこで、遺伝子破壊の為には、メダカが死なない最大濃度の変異誘導剤(ENU)で♂を処理し、大量の変異を精子DNAに誘導(飽和変異)したあと、♀と交配する。そして、たくさんのF1(5700匹)のDNAと精子とを凍結保管し、その5700匹のなかで、解析したい遺伝子が破壊された個体を、5700匹の各DNAをPCR→塩基配列決定によって捜す。
我々は、p53遺伝子破壊メダカを作製し、それが高発がんであることを確認した。p53は、大量のDNA損傷を持つ細胞に、アポプトーシスを誘導したり、複製をブロックすることによって、変異の直積を防止しているはずである。ただし、それを証明した研究はない。
この問題を解決する為に、我々は、野生型とp53欠損のメダカ♂を、それぞれENUで処理し、生存率を調べた。その結果、p53欠損メダカは野生型よりもENU耐性であることがわかった。これから、飽和変異♂と健康な♀から生じたF1の変異誘導率を解析予定である。

Research Products

• [Journal Article] Single stranded DNA binding protein hSSB1 is critical for genomic stability. (2008)
  • Author(s): Richard D, Bolderson E, Cubeddu L, Wadsworth RIM, Savage K, Sharma GG, Nicolette M.L, Tsvetanov S, Mcllwraith MJ, Pandita R, Takeda S, Hay TT, Gautier J, West SC, Paull TT, Pandital TK, White MF, Khanna KK
  • Journal Title: Nature (in press)
  • Peer Reviewed
• [Journal Article] DNA-Dependent Protein Kinase Inhibits AID-Induced Antibody Gene Conversion. (2007)
  • Author(s): Cook AJ, Raftery JM, Lau KK, Jessup A, Harris RS, Takeda S, Jolly CJ
  • Journal Title: PLoS Biol. 5 Pages: 80
  • Peer Reviewed
• [Journal Article] Cooperative Roles of Vertebrate Fbhl and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse. (2007)
  • Author(s): Kohzaki M, llatanaka A, Sonoda E, Yamazoe M, Kikuchi K, Trung NV, Szuts D, Sale JE, Shinagawa H, Watanabe M, Takeda S
  • Journal Title: Mol Cell Biol. 27 Pages: 2812-2820
  • Peer Reviewed
• [Journal Article] RAD18 and poly [ADP ribose]polymerase independently suppress the access of nonhomologous end joining to double strand breaks and facilitate homologous recombination mediated repair. (2007)
  • Author(s): Saberi A, Hochegger H, Szuts D, Lan L, Yasui A, Sale JE, Taniguchi Y, Murakawa Y, Hua W, Yokomori K, Helleday T, Teraoka H, Atakawa H, Buerstedde JM, Takeds S
  • Journal Title: Mol Cell Biol. 27 Pages: 2562-2571
  • Peer Reviewed
• [Journal Article] Interplay between DNA polymerases beta and lambda in repair of oxidation DNA damage in chicken DT40 cells. (2007)
  • Author(s): Tano K, Nakamura J, Asagoshi K, Arakawa H, Sonoda E, Braithwaite EK, Prasad R, Buerstedde JM, Takeda S, Watanabe M, Wilson SH
  • Journal Title: DNA Repair (Amst) 6 Pages: 869-875
  • Peer Reviewed
• [Journal Article] Histone H1 variant, H1R is involved in DNA damage response. (2007)
  • Author(s): Hashimoto H, Sonoda E, Takami Y, Kimura H, Nakayama T, Tachibana M, Takeda S, Shinkai Y
  • Journal Title: DNA Repair 6 Pages: 1584-1594
  • Peer Reviewed
• [Journal Article] Poly (ADP-ribose) Polymerase-1 Accelerates Single-Strand Break Repair in Concert with Poly (ADP-ribose) Glycohydrolase. (2007)
  • Author(s): Fisher A, Hochegger H, Takeda S, Caldecott KW
  • Journal Title: Mol Cell Biol. 27 Pages: 5597-5605
  • Peer Reviewed
• [Journal Article] An essential role for Cdkl in S phase control, revealed by chemical genetics in vertebrate cells. (2007)
  • Author(s): Hochegger H, Dejsuphong D, Sonoda E, Saberi A, Rajendra E, Kirk J, Hunt T, Takeda S
  • Journal Title: J Cell Biol. 178 Pages: 257-268
  • Peer Reviewed
• [Journal Article] Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells. (2007)
  • Author(s): Murakawa Y, Sonoda E, Barber LJ, Zeng W, Yokomori K, Kimura H, Niimi A, Lehmann AR, Zhao GY, Hochegger H, Boulton SJ and Takeda S
  • Journal Title: Cancer Res. 67 Pages: 8536-8543
  • Peer Reviewed
• [Journal Article] DNA damage-dependent acetylation and ubiquitination 1 of H2AX enhances chromatin dynamics. (2007)
  • Author(s): Ikura T, Tashiro S, Kakino A, Shima H, Jacob N, Amunugama R, Yoder K, Izumi S, Kuraoka I, Tanaka K, Kimura H, Ikura M, Nishikubo S, Ito T, Muto A, Miyagawa K, Takeda S, Fishel R, Igarashi K and Kamiya K
  • Journal Title: Mol Cell Biol. 27 Pages: 7028-7040
  • Peer Reviewed
• [Journal Article] Functional interactions between BLM and XRCC3 in the cell. (2007)
  • Author(s): Otsuki M, Seki M, Inoue E, Yoshimura A, Koto G, Yamanouchi S, Kawabe Y, Tada S, Shinohara A, Komura J, Ono T, Takeda S, Ishii Y, and Enomoto T
  • Journal Title: J Cell Biol. 179 Pages: 53-63
  • Peer Reviewed
• [Journal Article] Cells deficient in the FANC/BRCA pathway are hypersensitive to plasma levels of formaldehyde. (2007)
  • Author(s): Ridpath JR, Nakamura A, Tano K, Luke A.M., Sonoda E, Arakawa E, Buerstedde J-M, Gillespie DAF, Sale JE, Yamazoe M, Bishop DK, Takata M, Takeda S, Watanabe M, Swenberg JA, Nakamura J.
  • Journal Title: Cancer Res. 67 Pages: 11117-11122
  • Peer Reviewed
• [Journal Article] RAD51 up-regulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors. (2007)
  • Author(s): Martin RW, Orelli BJ, Yamazoe M, Minn AJ, Takeda S, Bishop DK
  • Journal Title: Cancer Res. 67 Pages: 9658-9665
  • Peer Reviewed
• [Journal Article] DDB1 gene disruption causes a severe growth defect and apoptosis in chicken DT40 cells. (2007)
  • Author(s): Wakasugi M, Matsuura K, Nagasawa A, Fua D, Shimizu H, Yamamoto K, Takeda S, Matsunaga T
  • Journal Title: Biochem Biophys Res Commun. 364 Pages: 771-777
  • Peer Reviewed
• [Journal Article] Ctp1/CtIP and the MRN complex collaborate in the initial steps of homologous recombination. (Review) (2007)
  • Author(s): Takeda S, Nakamura K, Taniguchi Y, Paull TT
  • Journal Title: Mol Cell 28 Pages: 351-352
  • Peer Reviewed
• [Presentation] BRCA1-CTIP complex plays a novel role in cellular tolerance to double-strand breaks associated with polypeptides in the presence of topoisomerase poisons. (2008)
  • Author(s): Takeda S, Nakamura K, Taniguchi Y
  • Organizer: Abcam, Maintenance of Genome Stability Conference 2008
  • Place of Presentation: Puerto Vallarta, Mexico
  • Year and Date: 20080304-07
• [Presentation] A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination (2007)
  • Author(s): Takeda S
  • Organizer: CNIO Cancer Conferences
  • Place of Presentation: Madrid, Spain,
  • Year and Date: 2007-11-06
• [Presentation] A critical role for the ubiquitin-conjugating enzyme UBC13 in initiating homologous recombination. (2007)
  • Author(s): Takeda S
  • Organizer: FASEB Genetic Recombination & Genome Rearrangements
  • Place of Presentation: Snowmass Village, Colorado, USA
  • Year and Date: 2007-07-29
• [Presentation] 遺伝子破壊メダカ作製系の樹立,淡路,兵庫,5月17日,2007.(招待、特別講演) (2007)
  • Author(s): 武田俊一
  • Organizer: 第5回幹細胞シンポジウム
  • Place of Presentation: 兵庫県淡路市
  • Year and Date: 2007-05-17