ヌクレオチド除去修復反応の細胞内調節機構に関する研究

• Project/Area Number: 12143202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kanazawa University
• Principal Investigator: 松永 司 金沢大学, 自然科学研究科, 教授 (60192340)
• Co-Investigator(Kenkyū-buntansha): 石垣 靖人 金沢大学, 自然科学研究科, 助手 (20232275) ; 若杉 光生 金沢大学, 自然科学研究科, 助手 (80345595) ; 二階堂 修 金沢大学, 薬学部, 教授 (60019669)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥31,600,000 (Direct Cost: ¥31,600,000); Fiscal Year 2004: ¥7,300,000 (Direct Cost: ¥7,300,000); Fiscal Year 2003: ¥7,300,000 (Direct Cost: ¥7,300,000); Fiscal Year 2002: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2001: ¥8,500,000 (Direct Cost: ¥8,500,000)
• Keywords: DNA損傷 / ヌクレオチド除去修復 / DDB / シクロブタン型ピリミジンダイマー / 紫外線 / ユビキチン・プロテアソーム系 / アポトーシス / c-Jun / 試験管内修復反応 / XPA / H2AX / (6-4)光産物 / 局所紫外線照射 / ゲルシフトアッセイ

Research Abstract

我々はこれまでDDB (DDB1/DDB2ヘテロダイマー)がヌクレオチド除去修復の促進因子として働くことを示してきたが、最近このDDBがユビキチン・プロテアソーム系にも関与することが示されており、本研究でもヌクレオチド修復の調節や修復以外におけるDDBの機能に視野を広げて解析を行った。具体的には、作製したDDB1特異的モノクローナル抗体を用いてDDB1の細胞内局在性を調べるとともに、DDB1とDDB2を各々ドキシサイクリン存在下で過剰発現する細胞株を樹立し、各サブユニットを過剰発現させたときの細胞への影響を検討した。その結果、抗DDB1モノクローナル抗体による免疫染色像は、主に細胞質に局在するという従来の過剰発現細胞を用いた報告とは異なり、核内でドット状の局在を示した。また、この染色像はDDB2遺伝子に変異をもつXP-E細胞、およびDDB2の発現が抑制されているチャイニーズハムスター細胞でも観察されることからDDB2に非依存的であることがわかり、他の因子の関与が考えられた。また、核の一部に局所紫外線照射を行うとこのドットはDNA損傷部位に集積し、この反応はDDB2に依存していた。一方、DDB1を過剰発現させた細胞ではドット状の染色像は見られず、多くは細胞質のみが強く均質に染色された。興味深いことに、このDDB1過剰発現細胞株は増殖能やコロニー形成能に著しい抑制が見られ、一部の細胞ではアポトーシス誘導が観察された。さらに、ヌクレオチド除去修復能には顕著な影響は認められなかったが、過剰発現時に細胞内のc-Jun量が顕著に増加し、またリン酸化体も増加していることがわかった。以上の結果より、DDB1がc-Junの活性調節およびアポトーシスにも関与する可能性が示唆された。

Research Products

• [Journal Article] Characterization of pathways dependent on the uvsde, uvrA1, or uvrA2 gene product for ultraviolet resistance in Deinococcus radiodurans. (2005)
  • Author(s): Tanaka, M.
  • Journal Title: J.Bacteriol. (In press)
• [Journal Article] Functional and physical interactions between ERCC1 and MSH2 for resistance to cis-platinum in mammalian cells. (2004)
  • Author(s): Lan, L.
  • Journal Title: DNA Repair 3(2) Pages: 135-143
• [Journal Article] Nuclear export signal in CDC25B. (2004)
  • Author(s): Uchida, S.
  • Journal Title: Biochem.Biophys.Res.Commun. 316(1) Pages: 226-232
• [Journal Article] Identification of the XPG region that causes onset of Cockayne syndrome using Xpg mutant mice generated by the cDNA-mediated knock-in method. (2004)
  • Author(s): Shiomi, N.
  • Journal Title: Mol.Cell.Biol. 24(9) Pages: 3712-3719
• [Journal Article] Binding of 14-3-3β but not 14-3-3σ controls the cytoplasmic localization of CDC25B : binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B. (2004)
  • Author(s): Uchida, S.
  • Journal Title: J.Cell Sci. 117(14) Pages: 3011-3020
• [Journal Article] Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen. (2004)
  • Author(s): Oyama, M.
  • Journal Title: Biochem.Biophys.Res.Commun. 321(1) Pages: 183-191
• [Publications] Fu, D.: "cDNA cloning of the chicken DDB1 gene encoding the p127 subunit of damaged DNA-binding protein."Gene Genet.Syst.. 78(2). 169-177 (2003)
• [Publications] Hirouchi, T.: "A gene for a Class II DNA photolyase from Oryza sativa : cloning of the cDNA by dilution-amplification."Mol.Gen.Genomics. 269(4). 508-516 (2003)
• [Publications] Sakamoto, A.: "Disruption of the AtREV3 gene causes hypersensitivity to ultraviolet B light and γ-rays in Arabidopsis : implication of the presence of a translesion synthesis mechanism in plants."Plant Cell. 15(9). 2042-2057 (2003)
• [Publications] Lan, L.: "Functional and physical interactions between ERCC1 and MSH2 for resistance to cis-platinum in mammalian cells."DNA Repair. 3(2). 135-143 (2004)
• [Publications] Uchida, S.: "Nuclear export signal in CDC25B."Biochem.Biophys.Res.Commun.. 316(1). 226-232 (2004)
• [Publications] Shiomi, N.: "Identification of the XPG region that causes onset of Cockayne syndrome using Xpg mutant mice generated by the cDNA-mediated knock-in method."Mol.Cell.Biol.. in press. (2004)
• [Publications] Wakasugi, M.: "DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair"J.Biol.Chem.. 277(3). 1637-1640 (2002)
• [Publications] Spivak, G.: "Ultraviolet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers"DNA Repair. 1(8). 629-643 (2002)
• [Publications] Fu, D.: "cDNA cloning of the chicken DDB1 gene encoding the p127 subunit of damaged DNA-binding protein"Gene Genet. Syst.. (in press). (2003)
• [Publications] Tanaka, M.: "Effects of pholoreactivation of cyclobutane pyrimidine dimers and pyrlimdine (6-4) pyriridone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichia coli"Mutagenesis. 16(1). 1-6 (2001)
• [Publications] Wakasugi, M.: "Damaged-DNA binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A"J. Biol. Chern.. 276(18). 15434-15440 (2001)
• [Publications] Katsumi, S.: "In situ visualization of ultraviolet light-induced DNA damage repair in locally irradiated human tibroblasts"J. Invest. Dermatol.. 117(5). 1156-1161 (2001)
• [Publications] Kiyosawa, K.: "Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant"Mutation Res.. 487(3-4). 149-156 (2001)
• [Publications] Mone, M.J.: "Local UV-induced DNA damage in cell nuclei results in local transcription inhibition"EMBO. Rep.. 2(11). 1013-1017 (2001)
• [Publications] Wakasugi, M.: "DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotideexcision repair"J. Biol. Chemn.. 277(3). 1637-1640 (2002)
• [Publications] Ohtoshi,E.: "Respective roles of cyclobutane pyrimidine dimers, (6-4) photoproducts and minor photoproducts in UV mutagenesis of repair deficient xeroderma pigmentosum A cells."Cancer Res.. 60(6). 1729-1735 (2000)
• [Publications] Torizawa,T.: "DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer."Nucleic Acids Res.. 28(4). 944-951 (2000)
• [Publications] Yokoyama,H.: "Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT (6-4) T photoproduct formed by ultraviolet radiation."J.Mol.Biol.. 299(3). 711-723 (2000)
• [Publications] Perdiz,D.: "Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis."J.Biol.Chem.. 275(35). 26732-26742 (2000)
• [Publications] Tanaka,M.: "Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichia coli."Mutagenesis. 16(1). 1-6 (2001)
• [Publications] Wakasugi,M.: "DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and RPA."J.Biol.Chem.. (in press). (2001)