ヌクレオチド除去修復による遺伝情報維持と細胞機能制御機構

• Project/Area Number: 12143207
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: 菅澤 薫 独立行政法人理化学研究所, 花岡細胞生理学研究室, 副主任研究員 (70202124)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥35,200,000 (Direct Cost: ¥35,200,000); Fiscal Year 2004: ¥8,700,000 (Direct Cost: ¥8,700,000); Fiscal Year 2003: ¥8,700,000 (Direct Cost: ¥8,700,000); Fiscal Year 2002: ¥8,800,000 (Direct Cost: ¥8,800,000); Fiscal Year 2001: ¥9,000,000 (Direct Cost: ¥9,000,000)
• Keywords: ヌクレオチド除去修復 / XPCタンパク質 / UV-DDB / ユビキチン化 / centrin 2 / 色素性乾皮症 / RAD23ホモログ / ユビキチン / プロテアソーム / DDB / DNA損傷認識 / XPC-HR23B複合体 / 塩基除去修復 / チミンDNAグリコシラーゼ / DNA損傷 / DNA損傷認識機構 / ピリミジン二量体 / コビキチン化

Research Abstract

細胞の紫外線照射に伴い、C群色素性乾皮症(XPC)タンパク質がUV-DDB依存的にユビキチン化されることを昨年までに明らかにした。細胞抽出液からの共免疫沈降実験、および精製タンパク質を用いたin vitroでの結合実験から、XPCとUV-DDBが直接物理的に相互作用することが示された。また、DDB1、DDB2に加えてcullin 4A、Roc1を昆虫細胞で同時に発現させることにより、ユビキチン・リガーゼ活性を持つUV-DDB-E3複合体を精製し、これを用いてXPCのユビキチン化反応を無細胞系で再構成することに成功した。この反応系ではXPCだけでなく、DDB2とcullin 4Aもユビキチン化の基質となるが、紫外線照射した細胞ではXPCのユビキチン化が可逆的であるのに対して、DDB2は速やかに分解されることがわかった。すなわち、XPCとDDB2は共通のE3によってユビキチン化されるにもかかわらず、その細胞内での運命が大きく異なることが明らかになった。
一方、XPCと複合体を形成するcentrin 2の機能を解析するため、まずXPCにおけるcebtrin 2結合領域を同定した。この結合領域に3か所のアミノ酸置換を導入することによってcentrin 2との結合能を欠く変異XPCを作成した。この変異XPCは細胞内、および無細胞系において、野生型XPCと比較してヌクレオチド除去修復における活性が有意に低下していた。精製タンパク質を用いた生化学的な解析により、centrin 2は複合体形成を介してXPCの損傷DNA結合活性を増強し、それによって修復反応を促進することが示された。

Research Products

• [Journal Article] Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex (2005)
  • Author(s): Yasuda, T. et al.
  • Journal Title: DNA Repair 4(3) Pages: 389-395
• [Journal Article] Nucleotide excision repair of 5-formyluracil in vitro is enhanced by the presence of mismatched bases (2004)
  • Author(s): Kino, K. et al.
  • Journal Title: Biochemistry 43(10) Pages: 2682-2687
• [Journal Article] Overproduction of eukaryotic SUMO-1- and SUMO-2-conjugated proteins in Escherichia coli (2004)
  • Author(s): Uchimura, Y. et al.
  • Journal Title: Analytical Biochemistry 331(1) Pages: 204-206
• [Journal Article] Relative levels of the two mammalian Rad23 homologs determine composition and stability of the xeroderma pigmentosum group C protein complex (2004)
  • Author(s): Okuda, Y. et al.
  • Journal Title: DNA Repair 3(10) Pages: 1285-1295
• [Publications] Janicijevic, A.et al.: "DNA bending by the human damage recognition complex XPC-HR23B."DNA Repair. 2. 325-336 (2003)
• [Publications] Ng, J.M.Y.et al.: "A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein."Genes and Development. 17. 1630-1645 (2003)
• [Publications] Fujiwara, K.et al.: "Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B."Journal of Biological Chemistry. 279. 4760-4767 (2004)
• [Publications] Araki, M., et al.: "Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins"Mutation Research. 459. 147-160 (2002)
• [Publications] Yokoi, M., et al.: "The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA"Journal of Biological Chemistry. 275. 9870-9875 (2000)
• [Publications] Yamane K., et al.: "Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity"Oncogene. 19. 1982-1991 (2000)
• [Publications] Winkler.G.S., et al.: "Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF"Biochemistry. 40. 160-165 (2001)
• [Publications] Sugasawa, K., et al.: "A multistep damage recognition mechanism for global genomic nucleotide excision repair"Genes and Development. 15. 507-521 (2001)
• [Publications] Kusumoto, R., et al.: "Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro"Mutation Research. 485. 219-227 (2001)
• [Publications] Araki, M., et al.: "Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair"Journal of Biological Chemistry. 276. 18665-18672 (2001)
• [Publications] Sugasawa, K., et al.: "A molecular mechanism for DNA damage recognition by the xeroderma pigmentosum group C protein complex"DNA Repair. 1. 95-107 (2002)
• [Publications] Ng, J.M.Y., et al.: "Developmental defects and male sterility in mice lacking the ubiquitin-like DNA repair gene mHR23B"Molecular and Cellular Biology. 22. 1233-1245 (2002)
• [Publications] Hey, T., et al.: "The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay"Biochemistry. 41. 6583-6587 (2002)
• [Publications] Uchida, A., et al.: "The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH"DNA Repair. 1. 449-461 (2002)
• [Publications] Shimizu, Y., et al.: "Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase"EMBO Journal. 22. 164-173 (2003)
• [Publications] Janicijevic, A., et al.: "DNA bending by the human damage recognition complex XPC-HR23B"DNA Repair. 2(in press). (2003)
• [Publications] Kusumoto, R. et al.: "Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro"Mutation Research. 485. 219-227 (2001)
• [Publications] Araki, M. et al.: "Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C protein complex that initiates global genome nucleotide excision repair"Journal of Biological Chemistry. 276. 18665-18672 (2001)
• [Publications] Sugasawa, K. et al.: "A molecular mechanism of DNA damage recognition by the xeroderma pigmentosum group C protein complex"DNA Repair. 1. 95-107 (2002)
• [Publications] Ng, J. M. Y. et al.: "Developmental defects and male sterility in mice lacking the ubiquitin-like DNA repair gene mHR23B"Molecular and Cellular Biology. 22. 1233-1245 (2002)
• [Publications] Sugasawa,K. et al.: "A multistep damage recognition mechanism for global genomic nucleotide excision repair"Genes & Development. 15(in press). (2001)
• [Publications] Kusumoto,R. et al.: "Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro"Mutation Research. (in press). (2001)
• [Publications] Winkler,G.S. et al.: "Novel functional interactions between nucleotide excision DNA repair proteins influencing the exzymatic activities of TFIIH,XPG, and ERCC1-XPF"Biochemistry. 40. 160-165 (2001)
• [Publications] Yamane,K. et al.: "Retinoblastoma susceptibility protein,Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity"Oncogene. 19. 1982-1991 (2000)