Molecular Mechanisms of DNA Damage Recoguition and Repair

• Project/Area Number: 08407072
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Osaka University
• Principal Investigator: HANAOKA Fumio Osaka Univ., IMCB,Professor, 細胞生体工学センター, 教授 (50012670)
• Co-Investigator(Kenkyū-buntansha): MAEKAWA Takafumi Osaka Univ.IMCB,Research Assistant, 細胞生体工学センター, 助手 (90273721) ; MASUTANI Chikahide Osaka Univ.IMCB,Research Assistant, 細胞生体工学センター, 助手 (40241252) ; OHKUMA Yoshiaki Osaka Univ.IMCB,Associate Professor, 細胞生体工学センター, 助教授 (70192515)
• Project Period (FY): 1996 – 1998
• Project Status: Completed (Fiscal Year 1998)
• Budget Amount: ¥37,600,000 (Direct Cost: ¥37,600,000); Fiscal Year 1998: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 1997: ¥9,200,000 (Direct Cost: ¥9,200,000); Fiscal Year 1996: ¥19,900,000 (Direct Cost: ¥19,900,000)
• Keywords: DNA repair / NER / xeroderma pigmentosum / XPC protein / Vpr protein / HHR23B protein / 26S proteasome / S5a protein / 修復 / ヌクレオチド除去修復 / RNAポリメラーゼII / XPE蛋白質 / ヌクレチオド除去修復 / TFIIH / XPG蛋白質 / p53蛋白質

Research Abstract

We previously isolated a protein complex which biochemically corrects the defect of XP-C cell extracts in nucleotide excision repair (NER) in vitro. In the present study, we have analyzed the functions of the protein complex, XPC/HHR23B, in NER in mammalian cells and obtained the following results.
1) In mouse cells, there are homologues of human XPC/HHR23B and another RAD23 homologue, HHR23A.They have quite high homologies with each other.
2) We found most of XPC, HHR23B and HHR23A in nucleus. They do not make a tight complex with other NER proteins.
3) HHR23B stimulates the NER activity of XPC protein in vitro.
4) HHR23A protein makes a complex with HIV-1 Vpr protein and affects the cell cycle progression.
5) A very limited region of HHR23B is necessary and sufficient for XPC binding as well as the stimulation of NER activity of XPC.
6) HHR23A could reconstitute a physical complex with XPC and stimulate the in vitro repair activity in place of HHR23B.
7) XPC/HHR23B complex works as the earliest damage detector to initiate global genome repair subpathway of NER.
8) We found that a component of 26S proteasome, S5a, associates with HHR23B in yeast two-hybrid system as well as in crude cell extracts.

Research Products

• [Publications] Sugasawa, K.: "HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro." Mol.Cell.Biol.16. 4852-4861 (1996)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] van der Spek, P.J.: "XPC and human homologs of RAD23:intracellular localization and relationship to other nucleotide excision repair complexes." Nucl.Acids Res.24. 2551-2559 (1996)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Withers-Ward, E.S.: "Human inbuanodeficieuly rirus type 1 Vpr interacts with HHR23A, acellalar proteia implicated in nucleotide excision DNA repair" J.Virol.71. 9732-9742 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Masutani, C.: "Identification and charactarization of XPC-biading domain of hHR23B" Mol.Cell.Biol.17. 6915-6923 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sugasawa, K.: "Two human homologues of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity" Mol.Cell.Biol.17. 6924-6931 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sugasawa, K.: "Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair" Mol.Cell. 2. 223-232 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sugasawa, K., Masutani, C., Uchida, A., Maekawa, T., van der Spek, P.J., Bootsma, D., Hoeijmakers, J.H., J., and Hanaoka, F.: "HHR23B,a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in uitro." Mol.Cell.Biol.16. 4852-4861 (1996)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] van der Spek, P.J., Eker, A., Rademakers, S., Visser, C., Sugasawa K., Masutani, C., Hanaoka, F., Bootsma, D., and Hoeijmakers, J.H.J.: "XPC and human homologs of RAD23 : intracellular localization and relationship to other nucleotide excision repair complexes." Nucl.Acids Res.24. 2551-2559 (1996)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Witheres-Ward, E.S., Jowett, J.B., Stewart, S.A., Xie, Y.M., Garfinkel, A., Shibagaki, Y., Chow, S.A., Shah, N., Hanaoka, F., Sawitz, D,G., Armstrong, R.W., Souza, L.M., and Chen, I.S.: "Human immunodeficiency virus type 1 Vpr interacts with HHR23A,a cellular protein implicated in nucleotide excision DNA repair." J.Virol.71. 9732-9742 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Masutani, C., Araki, M., Sugasawa, K., van der Spek, P.J., Yamada, A., Uchida, A., Maekawa, T., Bootsma, D., Hoeijmakers, J.H.J., and Hanaoka, F.: "Identification and characterization of XPC-binding domain of hHR23B." Mol.Cell.Biol.17. 6915-6923 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sugasawa, K., Ng, J.M.Y., Masutani, C., Maekawa, T., Uchida, A., van der Spek, P.J., Eker, A.P.M., Rademakers, S., Visser, C., Aboussekhra, A., Wood, R.D., Hanaoka, F., Bootsma, D., and Hoeijmakers, J.H.J.: "Two human homologues of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity." Mol.Cell.Biol.17. 6924-6931 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sugasawa, K., Ng, J.M.Y., Masutani, C., Iwai, S., van der Spek, P., Eker, A.P.M., Hanaoka, F., Bootsma, D., and Hoeijmakers, J.H.J.: "Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair." Mol.Cell.2. 223-232 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Eki,T.: "Mapping of the human genes encoding cyclin H(CCNH)and the CDK-activating kinase(CAK)assembly factor MATl(MNATl)to chromosome bands 5g13 3-g14 and 14g23." Genomics. 47. 115-120 (1998)
• [Publications] Carrier,F.: "Evidence for distinct Kinase-mediated pathways in gadd gene responses." Biochem.Pharmacol.55. 853-861 (1998)
• [Publications] Mizuno,T.: "The second largest subunit of the mouse DNA polymerased α-primase complex facilitates both the production and nudear translocation of the catalytic subunit of DNA polymerased." Mol.Cell.Biol.18. 3552-3562 (1998)
• [Publications] Kai,M.: "A new Drosophila ultraviolet light-damaged DNA recoguition and on nclease that selectively nicks a(6-4)photoproduct site." Biochim,Biophys.Acta. 1397. 180-188 (1998)
• [Publications] Okamoto,T.: "Analysis of the role of TFIIE in transcriptional regulation through structure-function studies of the TFIIEA subunit." J.Biol.Chem.273. 22254-22259 (1998)
• [Publications] Sugasawa,K.: "Xenoderuse pigmentsum group C protein complex is the initiator of global genome nucleotide excision repain." Mol.Cell. 2. 223-232 (1998)
• [Publications] Sakamoto,A.at al.: "Botle hypercrophic and dilated casdiomyopathies are caused by nutation of the same gane 5-sarooglycan,in hamstor:An dnimal wodel of disragted dystrophin-assoolated glycaprltein cemplax." Proc.Natl.Acad.Sci.USA. 94. 13873-13878 (1997)
• [Publications] Withors-Ward,E.S.et al.: "Human immunede ficioncy rirus type 1 Vpc interacts with HHR23A,a cellular protein implicated in nuclaetide excision DNA repair." J.Virol.71. 9732-9742 (1997)
• [Publications] Masutani,C.et al.: "Identification and charactoriration of XPC-binding domatu of hHR23B." Mol.Cell.Biol.17. 6915-6923 (1997)
• [Publications] Sugasawa,K.et al.: "Two humnau homologues of Rad23 are functionalty inturchangaable in complex farmation and scimulation of XPC repair activity." Mol.Cell.Biol.17. 6924-6931 (1997)
• [Publications] Sasaki,H.at al.: "Dependence of induction of interphase death of Chinase hamster ovary cells exposed to auslerated heany ious on linear energy traisfer." Radiation Ras.148. 449-454 (1997)
• [Publications] Ohkuma,Y.: "Multiple functions of general transcription factors TFIIE and TFIIH in transcription:Possible points of regulation by trans-acting factors." J.Biochem.122. 481-489 (1997)
• [Publications] Ikehata,H.et al.: "Incubation at the nonpermissive temparature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a tamperature-sensitive ubiquitin-activating enzyme E1." Mol.Cell.Biol.17. 1484-1489 (1997)
• [Publications] Osada,H.et al.: "Screening of cell cycle inhibitors from microbial metabolites by a bioassay using a mouse cdc2 mutant cell line tsFT210." Bioorg.Med.Chem.5. 193-203 (1997)
• [Publications] Mizuno,T.et al.: "Identification of the nuclear localization signal of mouse DNA priniase:nuclear transport of p46 subunit is facilitated by intaraction with p54 subunit." J.Cell.Sci.109. 2627-2636 (1996)
• [Publications] Ito,K.et al.: "C-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T antigen." EMBO J.15. 5636-5646 (1996)
• [Publications] Sugasawa,K.et al.: "HHR23B,a human Rad23 homolog,stimulatas XPC protein in nucleotide axcision repair in vitro." Mol.Cell.Biol.16. 4852-4861 (1996)
• [Publications] Van der Spek.P.J.et al.: "XPC and human homologs of RAD23:intracellular localization and relationship to other nucleotide axcision repair complexes." Nucl.Acids Res.24. 2551-2559 (1996)