転写とヌクレオチド除去修復機構を共役する新規蛋白質複合体の解析

• Project/Area Number: 12143203
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: 田中 亀代次 大阪大学, 大学院・生命機能研究科, 教授 (80144450)
• Co-Investigator(Kenkyū-buntansha): 西條 将文 大阪大学, 大学院・生命機能研究科, 助教授 (90221986) ; 倉岡 功 大阪大学, 大学院・生命機能研究科, 助手 (60335396) ; 竹内 聖二 大阪大学, 大学院・生命機能研究科, 助手 (10304065) ; 吉野 雅文 大阪大学, 大学院・生命機能研究科, 助手 (00346149)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥63,000,000 (Direct Cost: ¥63,000,000); Fiscal Year 2004: ¥14,500,000 (Direct Cost: ¥14,500,000); Fiscal Year 2003: ¥14,500,000 (Direct Cost: ¥14,500,000); Fiscal Year 2002: ¥17,000,000 (Direct Cost: ¥17,000,000); Fiscal Year 2001: ¥17,000,000 (Direct Cost: ¥17,000,000)
• Keywords: ヌクレオチド除去修復 / 転写と共役した修復 / コケイン症候群 / 紫外線高感受性症候群 / ユビキンチンリガーゼ / siRNA / ノックアウトマウス / 微小核融合法 / DNA損傷 / DNA修復 / 転写 / スプライシング / RNAポリメラーゼII / ユビキチン / WD40リピート / 色素性乾皮症 / ダブルノックアウトマウス / 核マトリクス / 小脳失調 / 蛋白質複合体 / 小脳発育 / アポトーシス / 遺伝子ターゲティング / ミスマッチ修復 / GTPase / 核移行

Research Abstract

転写と共役した修復(TCR)に関与するコケイン症候群A群(CSA)蛋白質は、DDB1、COP9 signalosome、Roc1、Cullin4Aと複合体を構成し、ユビキチン・リガーゼ(E3)活性を持つことを確認した。さらに、CSA蛋白質複合体は、紫外線照射した細胞ではRNA polymerase IIoとも結合した。他方、CS-A患者由来の変異CSA蛋白質では複合体の形成が起こらないこと、E3活性もないことを明らかにし、CSA複合体によるユビキチン化制御がTCR機構に関与することを強く示唆した。CSA蛋白質複合体に含まれるDDB1の役割を解明するため、DDB1遺伝子をコンデイショナルにノックアウトしたマウスの作成を行った。他方、転写およびTCRに関与するXAB2蛋白質複合体を精製し、6種類の構成因子からなることを明らかにした。siRNAでXAB2をノックダウンした細胞は、TCR能が低下し、紫外線高感受性となり、mRNAスプライシングにも異常を示した。XAB2が、TCR、転写、スプライシングに関与する多機能性蛋白質複合体の連結因子であることを示唆した。XAB2遺伝子をノックアウトしたマウスを作成し、ホモ接合体マウスは胚盤胞時期までに胎児致死となることを明らかにした。他方、TCRに異常をもつが、CSと異なり神経症状や早期老化徴候は示さず、軽微な皮膚症状のみを示すUV^s症候群の原因遺伝子座が10番染色体に存在することを明らかにした。さらに、このUV^s症候群が、10番染色体に座位するコケイン症候群B群(CSB)遺伝子のヌル突然変異によって発症していることを発見した。CSB遺伝子にヌル突然変異を持つにもかかわらず、この患者が何故CS徴候を示さないのかが疑問として残った。CS症候を強く示すCSB患者は短縮CSB蛋白質が生成されており、短縮CSB蛋白質が何らかの異常な機能を発揮しており、そのととがCS徴候の発症に関連している可能性を指摘した。

Research Products

• [Journal Article] Severe growth retardation and short life span of double mutant mice lacking Xpa and Exon 15 of Xpg. (2005)
  • Author(s): Naoko Shiomi, Masahiko Mori, Seiji Kito, Yoshi-Nobu Harada, Kiyoji Tanaka, Tadahiro Shiomi.
  • Journal Title: DNA Repair 4 Pages: 351-357
  • NAID: 130006999900
• [Journal Article] Disruption of mouse XAB2 gene involved in pre-mRNA splicing, transcription and transcription-coupled DNA repair results in preimplantation lethality. (2005)
  • Author(s): Rie Yonemasu, Mitsuyoshi Minami, Yoshimichi Nakatsu, Masayo Takeuchi, Isao Kuraoka, Yoichi Matsuda, Yujiro Higashi, Hisato Kondoh, Kiyoji Tanaka.
  • Journal Title: DNA Repair 4 Pages: 479-491
• [Journal Article] Blockage of transcription elongation by mammalian RNA polymearse II at cyclobutane pyrimidine dimer and 6-4 photoproduct. (2004)
  • Author(s): Joan Seah Mei Kwei, Isao Kuraoka.Katsuyoshi Horibata.Manabu Ubukata, Eiry Kobatake Shigenori Iwai.Hiroshi Handa, Kiyoji Tanaka
  • Journal Title: Biochemical and Biophysical Research Communications 320 Pages: 1133-1138
• [Journal Article] Inhibition of nucleotide excision repair by anti-XPA monoclonal antibodies which interfere with binding to RPA, ERCC1 and TFIIH. (2004)
  • Author(s): Masafumi Saijo., Toshiro Matsuda., Isao Kuraoka, Kiyoii Tanaka.
  • Journal Title: Biochemical and Biophysical Research Communications 321 Pages: 815-822
• [Journal Article] Complete absence of Cockayne syndrome group B gene product gives rise to UV-sensitive syndrome but not Cockayne syndrome. (2004)
  • Author(s): Katsuyoshi Horibata, Yuka Iwamoto, IsaoKuraoka, Nicolaas G.J.Jaspers, Masahiro Kurimasa, Mitsuo Oshimura, Masamitsu Ichihashi, Kiyoji Tanaka.
  • Journal Title: Proc.Natl.Acad.Sci., USA 101 Pages: 15410-15415
• [Journal Article] UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex.
  • Author(s): Kaoru Sugasawa, Yuki Okuda, Masafumi Saijo, Ryotaro Nishi, Noriyuki Matsuda, Gilbert Chu, Toshio Mori, Shigenori Iwai, Keiji Tanaka, Kiyoji Tanaka, Fumio Hanaoka.
  • Journal Title: Cell (in press.)
• [Publications] Isao Kuraoka: "Effects of endogeneous DNA base lesions on transcription elongation by mammalian RNA polymerase II : Implications for transcription-coupled DNA repair and transcriptional mutagenesis."J.Biological Chemistry. 278. 7294-7299 (2003)
• [Publications] Yan Ren: "Three novel mutations responsible for Cockayne syndrome group A."Genes & Genetic Systems. 78. 93-102 (2003)
• [Publications] Regina Groisman: "The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage."Cell. 113. 357-367 (2003)
• [Publications] Li Lan: "Functional and physical Interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells."DNA Repair. 3. 135-143 (2004)
• [Publications] Naoto Inukai: "A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis."J.Biological Chemistry. (in press).
• [Publications] Fumikazu Yamazaki: "XPA gene-deficient, SCF-transgenic mice with epidermal melanin are resistant to UV-induced carcinogenesis."J.Investigative Dermatology. (in press).
• [Publications] Minoru Ichikawa: "Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice"Mutation Research. 459. 285-298 (2000)
• [Publications] Yoshimichi Nakatsu: "XAB2, a novel tetratricopeptide repeat protein, involved in transcription-coupled DNA repair and transcription"J.Biol.Chem.. 275. 34931-34937 (2000)
• [Publications] Masahiko Nitta: "A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA"Nucleic Acid Research. 28. 4212-4218 (2000)
• [Publications] Machiko Murai: "Early postnatal ataxia and abnormal cerebellar development in mice lacking xeroderma pigmentosum group A and the Cockayne syndrome group B DNA repair genes"Proc.Natl.Acad.Sci., USA. 98. 13379-13384 (2001)
• [Publications] Shinya Kamiuchi: "Translocation of Cockayne syndrome group A protein to the nuclear matrix : possible relevance to transcription-coupled DNA repair"Proc.Natl.Acad.Sci., USA. 99. 201-206 (2002)
• [Publications] Masafumi Yoshino: "Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice"DNA Repair. 1. 935-940 (2002)
• [Publications] Hiroko Miyauchi-Hashimoto: "Carcinogen-induced inflammation and immunosuppression are enhanced in xeroderma pigmentosum group A (XPA) model mice associated with hyperproduction of prostaglandin E2"The Journal of Immunology. 166. 5782-5791 (2001)
• [Publications] Fumio Ide: "Xeroderma pigmentosum group A gene action as a protection factor against 4-nitroquinoline-1-oxide-induced tongue carcinogenesis"Carcinogenesis. 22. 567-572 (2001)
• [Publications] Kiyoji Tanaka: "UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency"Mutation Research. 477. 31-40 (2001)
• [Publications] Takatoshi Ishikawa: "Importance of DNA repair in carcinogenesis : evidence from transgenic and gene targeting studies"Mutation Research. 477. 41-49 (2001)
• [Publications] Machiko Murai: "Early postnatal ataxia and abnormal cerebellar development in mice lacking xeroderma pigmentosum group A and the Cockayne syndrome group B DNA repair genes"Proc.Natl.Acad.Sci., USA. 98. 13379-13384 (2001)
• [Publications] Shinya Kamiuchi: "Translocation of Cockayne syndrome group A protein to the nuclear matrix : possible relevance to transcription-coupled DNA repair"Proc.Natl.Acad.Sci., USA. 99. 201-206 (2002)
• [Publications] Minoru Ichikawa: "Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice"Mutation Research. 459. 285-298 (2000)
• [Publications] Hiroaki Murai: "Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice"Mutation Research. 450. 181-192 (2000)
• [Publications] Yoshimichi Nakatsu: "XAB2, a novel tetratricopeptide repeat protein, involved in transcription-coupled DNA repair and transcription"J.Biol.Chem.. 275. 34931-34937 (2000)
• [Publications] Masahiko Nitta: "A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA"Nucleic Acid Research. 28. 4212-4218 (2000)
• [Publications] Hiroko Miyauchi-Hashimoto: "Carcinogen-inducedn inflammation and immunosuppression are enhanced in xeroderma pigmentosum group A (XPA) model mice associated with hyperproduction of prostaglandin E2"The Journal of Immunology,. (in press).
• [Publications] Kiyoji Tanaka: "UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency"Mutation Research,. (in press).