哺乳類細胞における損傷乗り越えDNA複製の分子機構

• Project/Area Number: 12143204
• 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: 益谷 央豪 大阪大学, 大学院・生命機能研究科, 助教授 (40241252)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥31,100,000 (Direct Cost: ¥31,100,000); 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: ¥8,300,000 (Direct Cost: ¥8,300,000); Fiscal Year 2001: ¥8,200,000 (Direct Cost: ¥8,200,000)
• Keywords: 色素性乾皮症バリアント / 損傷乗り越えDNA複製 / DNAポリメラーゼ / DNA損傷 / 突然変異 / 色素性乾皮症 / XPV / translesion synthesis / DNA polymerase η / DNA polymeraseι / DNAポリメラーゼη / 忠実度

Research Abstract

高発癌性の遺伝疾患のひとつである色素性乾皮症バリアント(XP-V)群の患者では、損傷DNAの複製機構に異常がある。本申請者は、XPV責任遺伝子産物であるDNAポリメラーゼηを同定し、これが、紫外線損傷DNAを鋳型として複製を行えるDNA合成酵素であることを明らかにし、ヒトにおける損傷DNAに対する防御機構のひとつとして損傷乗り越えDNA複製機構が重要な役割を担っていることを明らかにし、損傷乗り越えDNA複製研究という分野を切り開いてきた。これまでの研究の中で、本分野における重要な問題点として、DNAポリメラーゼηによるDNA合成の忠実度は本質的に低いものであり、その反応を損傷DNAの乗り越え複製に限局するメカニズムがあるであろうこと、また、DNAポリメラーゼηと類似したDNAポリメラーゼが複数存在し、これらを制御するメカニズムがあるであろうことなどを示してきた。本年度は、特に、これらの問題点に対して顕著な成果を挙げた。具体的には、DNAポリメラーゼηのDNA結合能について解析を行い、DNAポリメラーゼηが損傷に対して正しいヌクレオチドを重合することによりDNAポリメラーゼηとDNAとの結合が安定化されること、この安定化は重合されたヌクレオチドが正しい場合にのみ認められ、誤ったヌクレオチドを重合した場合には認められないこと、また、この安定化は、損傷を通過して二つ目のヌクレオチドを重合するまで維持されること、そして、このふたつめのヌクレオチドが重合されることにより複製型のDNAポリメラーゼが伸長反応を行えるようになることを示した。これらの結果より、DNAポリメラーゼη自身のDNA結合能により、DNAポリメラーゼηの反応が必要最小限に限定されていることが示された。一方で、DNAポリメラーゼηと他のタンパク質問の相互作用について検討し、DNAポリメラーゼηとREV1との間に物理的な相互作用を見出した。REV1はDNAポリメラーゼηと同じくYファミリーに分類されるDNA合成酵素であり、この相互作用が損傷乗り越えDNAポリメラーゼの使い分けにおいて重要と考えられる。

Research Products

• [Journal Article] Palm mutants in DNA polymerases α and η alter DNA replication fidelity and translesion activity. (2004)
  • Author(s): Niimi, A., et al.
  • Journal Title: Molecular and Cellular Biology 24 Pages: 2734-2746
• [Journal Article] Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer. (2004)
  • Author(s): Takasawa, K., Masutani, C., et al.
  • Journal Title: Nucleic Acids Research 32 Pages: 1738-1745
• [Journal Article] Translesion synthesis past estrogen-derived DNA adducts by human DNA polymerases η and κ. (2004)
  • Author(s): Suzuki, N., et al.
  • Journal Title: Biochemistry 43 Pages: 6304-6311
• [Journal Article] Interaction of hREVl with three human Y-family DNA polymerases. (2004)
  • Author(s): Ohashi, E., et al.
  • Journal Title: Genes to Cells 9 Pages: 523-531
• [Journal Article] High-efficiency bypass of DNA damage by human DNA polymerase Q. (2004)
  • Author(s): Seki, M., Masutani, C., et al.
  • Journal Title: EMBO Journal 23 Pages: 4484-4494
• [Journal Article] DNA binding properties of human DNA polymerase η : Implication for polymerase switching. (2004)
  • Author(s): Kusurnoto, R., Masutani, C., et al.
  • Journal Title: Genes to Cells 9 Pages: 1139-1150
• [Publications] Shimizu, M., et al.: "Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases."EMBO Reports. 4. 269-273 (2003)
• [Publications] McDonald, J.P., et al.: "129-derived strains of mice are deficient in DNA polymerase ι and have normal immunoglobulin hypermutation."J.Exp.Med.. 198. 635-643 (2003)
• [Publications] Jaloszynski, P., Masutani, C., et al.: "8-Hydroxyguanine in a mutational hotspot of c-Ha-ras gene causes misreplication, "action-at-a-distance" mutagenesis and inhibition of replication."Nucleic Acids Res.. 31. 6085-6095 (2003)
• [Publications] Higurashi, M., et al.: "Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA"J.Biol.Chem.. 278. 51968-51973 (2003)
• [Publications] Bassett, E., et al.: "Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases β and η in vitro."Biochemistry. 42. 14197-14206 (2003)
• [Publications] Niimi, A., et al.: "Palm residue mutants in DNA polymerases α and η alter DNA replication fidelity and translesion activity."Mol.Cell.Biol.. (In press).
• [Publications] Shimizu, M., et al.: "Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases"EMBO Reports. 4. 269-273 (2003)
• [Publications] Bassett, E., et al.: "Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerase β and η"DNA Repair. 1. 1003-1016 (2002)
• [Publications] Yamada, A., et al.: "Detection of reduced RNA synthesis in UV-irradiated Cockayne syndrome group B cells using an isolated nuclear system"Biochim.Biophys.Acta. 1592. 129-134 (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] Kusumoto, R., et al.: "Translesion synthesis by human DNA polymerase η across thymine glycol lesions"Biochemistry. 41. 6090-6099 (2002)
• [Publications] Chiapperino, D., et al.: "Preferential misincorporation of purine nucleotides by human DNA polymerase η opposite benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts"J.Biol.Chem.. 277. 11765-11771 (2002)
• [Publications] Chiapperino, D. et al.: "Preferential misinocorporation of purine nucleotides by human DNA polymerase η opposite benzo[a]pyrene 7.8-diol 9.10-epoxide deoxyguanosine adducts"J.Biol.Chem.. (in press). (2002)
• [Publications] Broughton, B.C. et al.: "Molecular analysis of mutations in DNA polymerase of in xeroderma pigmentosum variant patients"Proc.Natl.Acad.Sci.USA. 99. 815-820 (2002)
• [Publications] Kuraoka, I. et al.: "Oxygen free-radical damage to DNA : translesion synthesis by human DNA polymerase η and resistance to exonuclease action at cyclopurine deoxynucleaside residues"J.Biol.Chem.. 276. 49283-49288 (2001)
• [Publications] Matsuda, T. et al.: "Error rate and specificity of human and murin DNA polymerase η"J.Mol.Biol.. 312. 335-346 (2001)
• [Publications] Levine, R.L. et al.: "Translesion DNA synthesis catalyzed by human pol η and pol κ across 1,N^6-ethenodeoxyadenosine"J.Biol.Chem.. 276. 18717-18721 (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"J.Biol.Chem.. 276. 18665-18672 (2001)
• [Publications] Ishikawa, T. et al.: "Mutagenic and non-mutagenic bypass of DNA lesions by Drasophila DNA polymerases dpolη and dpol i"J.Biol.Chem.. 276. 15155-15163 (2001)
• [Publications] Ura, K.et al.: "ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes"EMBO J.. 20. 2004-2014 (2001)
• [Publications] Kusumoto, R. et al.: "Diversity of the damage recognition step in the global genomic nucleotide exicision repair in vitro"Mutation Res.. 485. 219-227 (2001)
• [Publications] Sugasawa, K. et al.: "A multistep damage recognition mechanism for global genomic nucleotide excision repair"Genes Dev.. 15. 507-521 (2001)
• [Publications] Bebenck, K. et al.: "Proofreading of DNA polymerase η-dependent replication errors"J.Biol.Chem.. 276. 2317-2320 (2001)
• [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 (2000)
• [Publications] Yokoi,M. et al.: "The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of TFIIH to damaged DNA."J.Biological Chemistry. 275. 9870-9875 (2000)
• [Publications] Vaisman,A. et al.: "Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase η."Biochemistry. 39. 4575-4580 (2000)
• [Publications] Matsuda,T. et al.: "Low fidelity DNA synthesis by human DNA polymerase η."Nature. 404. 1011-1013 (2000)
• [Publications] Watanabe,Y.et al.: "Modulation of TFIIH-associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II."Genes to Cells. 5. 407-423 (2000)
• [Publications] Masutani,C. et al.: "Mechanisms of accurate translesion synthesis by human DNA polymerase η."EMBO J.. 19. 3100-3109 (2000)
• [Publications] Yamada,A. et al.: "Complementation of defective translesion synthesis and ultraviolet light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase η."Nucleic Acids Research. 28. 2473-2480 (2000)
• [Publications] Ohashi,E. et al.: "Erron-prone bypass of certain DNA lesions by the human DNA polymerase κ."Genes and Development. 14. 1589-1594 (2000)
• [Publications] Miao,F. et al.: "3-methyladenine-DNA glycosylase (MPG protein) interacts with human RAD23 proteins"J.Biological Chemistry. 275. 28433-28438 (2000)
• [Publications] Yuasa,M. et al.: "Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene."Oncogene. 19. 4721-4728 (2000)
• [Publications] Bebenek,K. et al.: "Proofreading of DNA polymerase η-dependent replication errors."J.Biological Chemistry. 276. 2317-2320 (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] Ishikawa,T. et al.: "Mutagenic and non-mutagenic bypass of DNA lesions by Drosophila DNA polymerases dpal η and dpoli."J.Biological Chemistry. (in press). (2001)
• [Publications] Araki,M. et al.: "Centrosame protein centrin Z/cottractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair."J.Biological Chemistry. (in press). (2001)
• [Publications] Ura,K. et al.: "ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes."EMBO J.. (in press). (2001)
• [Publications] Levine,R.L. et al.: "Translesion DNA synthesis catalized by human pol ηand polκ across 1.N^6-ethenodeoxyadenosine."J.Biological Chemistry. (in press). (2001)