Structural Basis for mechanisms of preventing genomic instability

• Project/Area Number: 15390016
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: KUMAMOTO UNIVERSITY
• Principal Investigator: YAMAGATA Yuriko KUMAMOTO UNIVERSITY, FACULTY OF MEDICAL AND PHARMACEUTICAL SCIENCES, PROFESSOR, 大学院・医学薬学研究部, 教授 (40183678)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥14,400,000 (Direct Cost: ¥14,400,000); Fiscal Year 2004: ¥4,800,000 (Direct Cost: ¥4,800,000); Fiscal Year 2003: ¥9,600,000 (Direct Cost: ¥9,600,000)
• Keywords: Preventing genomic instability / Enzyme-substrate interaction / Protein Crystallography / Structural biology / Bio-molecular interaction / 蛋白質-リガンド相互作用 / DNA複製 / 蛋白質X線結晶構造解析

Research Abstract

Cellular DNAs, RNAs and Nucleotides are remarkable instable. Therefore cells have varieties of preventing systems for genomic instability. In this study, we target the proteins which work during pre-, first or middle stage of DNA replication. Our research purpose is to elucidate the preventing mechanisms for the genomic instability at the atomic level by the determinations of these protein structures.
The structures of MutT and hMTH1 were determined in the complexes with substrates and products by X-ray crystallography. The crystal structures of MutT alone, and in complex with a product, 8-oxo-dGMP(MutT-8-oxo-dGMP) have shown that MutT specifically recognizes 8-oxo-dGMP through a wealth of hydrogen bonds to the protein and waters in the binding pocket with the large ligand-induced conformational change. The mammalian counterpart of MutT, MutT homolog-1(MTH1), can hydrolyze a variety of nucleoside triphosphates containing oxidized adenine. The structural basis for the difference of the substrate specificity between MutT and hMTH1 is of fundamental interest. The crystal structure of MTH1 complexed with a product, 8-oxo-dGMP(hMTH1-8-oxo-dGMP) shows that the means of 8-oxoG recognition by hMTH1 are different from those found in the structure of MutT-8-oxo-dGMP. The structure of the substrate-binding pocket in hMTH1-8-oxo-dGMP suggests that hMTH1 can recognize several oxidatively damaged purine nucleoside triphosphates without a large conformational change.
The crystals of N-terminally His-tagged NUDT5 grew, but the resolution of the diffraction was relatively low. Now we are trying to get the crystals diffracting to a higher resolution by cutting the His-Tag.
The crystallization trials of purified primase and AlkB were in progress.

Research Products

• [Journal Article] Identification of single Mn2+ binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography (2005)
  • Author(s): Tsunaka Y. et al.
  • Journal Title: J.Mol.Biol. 345 Pages: 1171-1183
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification of single Mn^<2+> binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography. (2005)
  • Author(s): Tsunaka, Y., Takano, K., Matsumura, H., Yamagata, Y., Kanaya, S.
  • Journal Title: J.Mol.Biol. 345 Pages: 1171-1183
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency (2004)
  • Author(s): Yoshioka Y. et al.
  • Journal Title: Biochem.Biophys.Res.Commun. 318 Pages: 808-814
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates (2004)
  • Author(s): Mishima M. et al.
  • Journal Title: J.Biol.Chem. 279 Pages: 33806-33815
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex forms (2004)
  • Author(s): Nakamura T. et al.
  • Journal Title: Acta Cryst. D60 Pages: 1641-1643
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Functionalization of TNF-alpha using phage display technique and PEGylation improves its antitumor therapeutic window (2004)
  • Author(s): Shibata H. et al.
  • Journal Title: Clinical Cancer Res. 10 Pages: 8293-8300
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Optimal site-specific PEGylation of mutant TNF-α improves its antitumor potency (2004)
  • Author(s): Y.Yoshioka, Y.Tsutsumi, S.Ikemizu, Y.Yamamoto, H.Shibata, T.Nishibata, Y.Mukai, M.Okamoto, M.Taniai, T.Shimizu, M.Kawamura, Y.Abe, S.Nakagawa, S.Nagata, Y.Yamagata, T.Mayumi
  • Journal Title: Biochem Biophys Res Commun. 315 Pages: 808-814
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The Crystal Structure of the Tryptophan Synthase β Subunit from the Hyperthermophile, Pyrococcus furiosus : Investigation of Stabilization Factors. (2004)
  • Author(s): Hioki, Y., Ogasahara, K., Lee, S., Ma, J., Ishida, M., Yamagata, Y., Matsuura, Y., Ota, M., Ikeguchi, M., Kuramitsu, S., Yutani, K.
  • Journal Title: Eur.J.Biochem. 271 Pages: 2624-2635
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Antiproferative Constituents on Plants 14. Coumarins and Acridone Alkaloids from Boenninghausenia japonica Nakai. (2004)
  • Author(s): Chaya, N., Terauchi, K., Yamagata, Y., Kinjo, J., Okabe, H.
  • Journal Title: Biol.Pharm.Bull. 27 Pages: 1312-1316
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates. (2004)
  • Author(s): Mishima, M., Sakai, Y., Itoh, N., Kamiya, H., Furuichi, M., Takahashi, M., Yamagata, Y., Iwai, S., Nakabeppu, Y., Shirakawa, M.
  • Journal Title: J.Biol.Chem. 279 Pages: 33806-33815
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex forms. (2004)
  • Author(s): Nakamura, T., Doi, T., Sekiguchi, M., Yamagata, Y.
  • Journal Title: Acta Cryst. D60 Pages: 1641-1643
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Functionalization of TNF-α using phage display technique and PEGylation improves its antitumor therapeutic window. (2004)
  • Author(s): Sibata, H., Yoshioka, Y., Ikemizu, S., Kobayashi, K., Yamamoto, Y., Mukai, Y., Okamoto, T., Taniai, M., Kawamura, M., Abe, Y., Nakagawa, S., Nagata, S., Yamagata, Y., Mayumi, T., Tsutsumi, Y.
  • Journal Title: Clinical Cancer Res. 10 Pages: 8293-8300
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The Crystal Structure of the Tryptophan Synthase β Subunit from the Hyperthermophile, Pyrococcus furiosus
  • Author(s): Hioki Y. et al.
  • Journal Title: Eur.J.Biochem. 271 Pages: 2624-2635
  • Description: 「研究成果報告書概要(和文)」より
• [Book] タンパク質のかたちから生命の謎を解く (2005)
  • Author(s): 田之倉優 編
  • Total Pages: 232
  • Publisher: 株式会社 クバプロ
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nakano H. et al.: "Structure and binding mode of a ribosome recycling factor (RRF) from mesophilic bacterium"Journal of Biological Chemistry. 278. 3427-3436 (2003)
• [Publications] Takano K. et al.: "Buried Water Molecules Contribute to the Conformational Stability of a Protein"Protein Engineering. 16. 5-9 (2003)
• [Publications] Takatsuna H. et al.: "Identification of TIFA as an adapter protein that links TRAF6 to IRAK-1 in IL-1 receptor signaling"Journal of Biological Chemistry. 278. 12144-12150 (2003)
• [Publications] Yoshioka Y. et al.: "Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency"Biochemical and Biophysical Research Communications. 315. 808-814 (2004)
• [Publications] 加藤昭夫ら著: "タンパク質工学"株式会社医学出版. 305 (2003)