生体金属による遺伝子発現制御

• Project/Area Number: 08249103
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: Hiroshima University
• Principal Investigator: 木村 栄一 (木村 榮一) 広島大学, 医学部, 教授 (30034010)
• Co-Investigator(Kenkyū-buntansha): 石浜 明 国立遺伝学研究所, 分子遺伝研究系, 教授 (80019869) ; 杉浦 幸雄 京都大学, 化学研究所, 教授 (40025698) ; 塩谷 光彦 岡崎国立共同研究機構, 分子科学研究所, 教授 (60187333) ; 青木 伸 広島大学, 医学部, 助手 (00222472)
• Project Period (FY): 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥121,100,000 (Direct Cost: ¥121,100,000); Fiscal Year 1999: ¥18,700,000 (Direct Cost: ¥18,700,000); Fiscal Year 1998: ¥32,300,000 (Direct Cost: ¥32,300,000); Fiscal Year 1997: ¥44,300,000 (Direct Cost: ¥44,300,000); Fiscal Year 1996: ¥25,800,000 (Direct Cost: ¥25,800,000)
• Keywords: 大環状ポリアミン / DNaseI / 亜鉛錯体 / Znフィンガー / 塩基配列認識 / プロモーターDNA / TATAボックス / RNAポリメラーゼ / 人工β-cヌクレオチド / 亜鉛フィンガー / 生体金属 / 遺伝子発現制御 / Zn-フィンガー / 亜鉛 / 核酸塩基 / 転写因子 / 抗HIV活性 / 遺伝子プロモーター領域 / DNA融解温度

Research Abstract

1.核酸-合成金属錯体相互作用の研究:
12員環テトラアミン(cyclen)の亜鉛錯体が、核酸塩基中イミド基をもつチミン(又はウラシル)だけを選択的に認識しComplexをつくることを発見したが、それを大きく展開し、DNA(例:大腸菌プラスミドDNA(150bp))中のTと選択的に結合することをDNaseI及びmicrococcal nuclease footprint法により証明した。更に、Zn^<2+>-cyclen錯体は、DNA TATAboxに選択的に結合し、転写因子TFIIDの活性化機構を抑え、今までに例のない全く新しいタイプの遺伝子発現制御分子となることが明らかとなった。また、それらの化学的、生化学的機構の結果、抗菌活性を示すことも発見した。(木村栄一)
2.蛋白-核酸相互作用に関与する生体金属の研究:
転写因子Spl中のZnフィンガー(転写因子)Cys_2-His_2型亜鉛配位構造motifを3倍に拡大した新たな9つのZn-フィンガーからなるペプチドを合成し、DNAの30塩基対配列を選択的に認識する分子を創造した。また、二つのSplDNA結合ドメインを適当な長さのグリシンポリペプチドでlinkした分子を合成し、それらがDNAをおれ曲げることを発見、新たな遺伝子発現制御小工分子となりうることを示した。(杉浦幸雄)
(Fe^<2+>-EDTA)を大腸菌RNAポリメラーゼのCysやLysに共有結合させる方法を考案し、接触相手の転写因子蛋白やDNAとの接点部固定した。RNAポリメラーゼのシグマサブユニットのC端ドメインがDNAエンハンサー部と接触すること、サブユニット間の接点、シグマサブユニットのプロモーターDNAとの接点、多数の転写因子のポリメラーゼ上の接点等も明らかとなった。FeのO_2との反応を利用した分子間相互作用を解析する本法は、転写研究に新たなbreakthroughをもたらしたのみならず、他成分分子集合体や、多分子相互作用が主要な研究対象となって来た現代生命科学で、大変有用なtoolとなろう。(石浜明)

Research Products

• [Publications] E.Kikuta et al.: "Novel Recognition of Thymine Base in Double-Stranded DNA by Zinc(II)-Macrocyclic Tetraamine Complexes Appended with Aromatic Groups"J.Am.Chem.Soc.. 121(23). 5426-5436 (1999)
• [Publications] H.Kurosaki et al.: "New Robust Bleomycin Analogues : Synthesis, Spectroscopy, and Cristal Structures of the Copper(II) Complexes"Inorg.Chem.. 38. 2824-2832 (1999)
• [Publications] E.Kikuta et al.: "Natural and synthetic double-stranded DNA binding studies of macrocyclictetraamine Zinc(II) complexes appended with polyaromatic groups"J.Biol.Inorg.Chem.. 4. 431-440 (1999)
• [Publications] E.Kimura et al.: "Selective and Efficient Recognition of Thymidylylthymidine (TpT) by Bis(Zn^<2+>-cyclen) and Thymidylythymidylythymidine(TpTpT) by Tris(Zn^<2+>-cyclen) at Neutral pH in Aqueous Solution"Chem.Eur.J.. 5(11). 3113-3123 (1999)
• [Publications] H.Kurosaki et al.: "Synthesis, spectroscopic, redox properties, and DNA cleavage activity of low-spin iron(III) complexes of bleomycin analogues"Inorg.Chimica.Acta.. 294. 56-61 (1999)
• [Publications] S.Aoki et al.: "Three-Dimensional Supermolecules Assembled from a Tris(Zn^<2+>-cyclen)Complex and Di- and Trianionic Cyanuric Acid in Aqueous Solution (Cyclen=1, 4, 7, 10-Tetraazacyclododecane)"J.Am.Chem.Soc.. 122(4). 576-584 (2000)
• [Publications] Y.Sugiura et al.: "Comparison of the Sequence-Selective DNA Binding by Peptide Dimers with Covalent and Noncovalent Dimerization Domains"Biochemistry. 38. 1626-1632 (1999)
• [Publications] Y.Sugiura et al.: "Fluorescence Property of Oxazole Yellow-linked Oligonucleotide : Triple Helix Formation and Photocleavage of Double-standed DNA in the Presence of Spermine"Bioog.Med.Chem.. 7. 1207-1211 (1999)
• [Publications] Y.Sugiura et al.: "Fluorescence Property of Oxazole Yellow-linked Oligonucleotide : Triple Helix Formation and Photocleavage of Double-standed DNA in the Presence of Spermine"Bioog.Med.Chem.. 7. 1207-1211 (1999)
• [Publications] A.Ishihama et al.: "Organisation of open complexes at Escherichia coli promoters : mapping the promoter DNA sites close to region 2.5 of the σ^<70> subunit of RNA polymerase"J.Biol.Chem.. 274. 2263-2270 (1999)
• [Publications] 木村栄一: "The First Selective and Efficient Transport of Imide-Containing Nucleosides and Nucleotides by Lipophilic Cyclen-Zinc (II) Complexes." J.Am.Chem.Soc.120. 10018-10026 (1998)
• [Publications] 木村栄一: "Efficient Inhibition of Photo [2+2] cycloaddition of Thymidilyl (3'-5') Thymidine and Promotion of Photosplitting of the cis-syn-Cyclobutane Thymine Dimer by Dimeric Zinc (II) -Cyclen Complexes Containing m-and p-Xylyl Spacers." J.Am.Chem.Soc.120. 10094-10102 (1998)
• [Publications] 木村栄一: "Dynamic Anion Recognition by Macrocyclic Polyamines in Neutral pH Aqueous Solution : Development from Static Anion Complexes to an Enolate Complex." J.Chem.Soc.Chem.Comm.1495-1500 (1998)
• [Publications] 杉浦幸雄: "Artificial Nine Zinc-Finger Peptide with 30 Base pair Binding Sites" Biochenistry. 37. 13827-13834 (1998)
• [Publications] 塩谷光彦: "Macrocyclic Zinc (II) Complexes for Selective Recognition of Nucleobases in Single- and Double-Stranded Polynucleotides." J.Bioimorg.Chem.3. 259-267 (1998)
• [Publications] 石浜明: "Organisation of open complexes at Escherichia coli promoters : mapping the promoter DNA sites close to region 2.5 of the σ^<70> subunit of RNA polymerase." J.Biol.Chem.274. 2263-2270 (1999)
• [Publications] Eiichi Kimura: "Structure and O_2 uptake preperties of a novel nickel (II) wmplex of pyridyl-pendant dioxocyclam [1-(2-pyridyl) methyl-5,7-dioxo-1,4,8,11-tetraazacyclo tetradecane" J.Biol.Inorg.Chem.2・1. 74-82 (1997)
• [Publications] Eiichi Kimura: "A tris(Zn^<II>-1,4,7,10-tetraazacyclodade cannel complex as a new receptor for phosphate dianions in aqueous solution" J.Am.Chem.Soc.119・13. 3068-3076 (1997)
• [Publications] Xian He Bu: "Crystal structure and properties of a new copper^<(II)> wmplex of dioxocyclam appended with 8-methyl quinolire (Dioxocyclam = 1,4,8,11-tetraazacyclotetradecane-5,7-dione)" J.Incul.Phenom.Mol.Recog.Chem. 27.3. 245-258 (1997)
• [Publications] Eiichi Kimura: "Fcile and selective electrostatic stabilization of urocil N (1) -anion by a proximale protonated amine : An implication for why urocil N (1) is chosen by glycosylazien site" J.Am.Chem.Soc.119・45. 10909-10919 (1997)
• [Publications] Takashi Morii: "Factors govering the sequence-selective DNA binding of geometrically constrained peptide dimers" J.Am.Chem.Soc. 119・16. 3649-3655 (1997)
• [Publications] Yukio Sugiura: "Selective cleavage of tRNAphe with secondary and tertiary structures by enedyne antitumor antibiotics" Bioorg.Med.Chem. 5・6. 1229-1234 (1997)
• [Publications] Eiichi Kimura: "Metal Sites in Proteins and Models Phosphatases,Lewis Acids and Vanadium" Springer-Verlag, 28 (1997)
• [Publications] H. Fujioka et. al.: "A new Bis (Zinc (II) -Cyclen) Complex as a Novel Chelator for Barbiturates and Phosphates" Heterocycles. 42 (2). 775-787 (1996)
• [Publications] E. Kimura et. al: "Zinc Complexes as Targeting Agents for Nucleic Acids" "Metal Ions in Biological Systems" ed. by A. Sigel and H. Sigel. Marcel Dekker. Inc.33. 29-52 (1996)
• [Publications] Y. Inouye et. al: "Differential Contribution of Metal Complexation and Dimerization to the Chemotherapeutic Potential of Bicyclen-Zn2^<II> Complex against Human Immunodeficiency Virus" Biol. Pharm. Bull.19 (3). 456-458 (1996)
• [Publications] T. Koike et. al: "Novel Properties of Cooperative Dinuclear Zinc (II) Ions : The Selective Recognition of Phosphomonoesters and Their P-O Ester Bond Cleavage by a New Dinuclear Zinc (II) Cryptate" J. Am. Chem. Soc. 118. 3091-3099 (1996)
• [Publications] T. Koike et. al: "Bis (Zn^<II>-Cyclen) Complex as a Novel Receptor of Barbiturates in Aqueous Solution." Chem. Europ. J. 2. 617-623 (1996)
• [Publications] X. H. Bu et.al: "Stabilization of Nickel (III) ion by New Macrocyclic Diozotetraamines Bearing Functional Pendant Donors" Polyhedron. 15. 161-163 (1996)
• [Publications] Y. Kodama et. al: "A New Cyclam with a Triphenylphosphine-pendant and its Metal Complexes" Inorg. Chim. Acta.246. 151-158 (1996)
• [Publications] T. Koike et. al: "Metalloenzyme Models that Answer Mysteries Surrounding the Intrinsic Properties of Zn" Comprehensive Supramoleculaar Chemistry' 10, Pergamon. 429-444 (1996)
• [Publications] E. Kimura: "Creation of new Supramoleculaar Science with Macrocyclic Polyamines" (1996 Pharmaceutical Society of Japan Award Review) Yakugaku Zasshi. 116. 587-605 (1996)
• [Publications] H. Hashimoto et. al: "Hydrolysis of Lipophilic Esters Catalyzed by a Zinc (II) Complex of a Long Alkyl-Pendant Macrocyclic Teraamine in Micellar Solution" J. Am. Chem. Soc.118. 10963-10970 (1996)
• [Publications] X. H. Bu et. al: "Synthesis, Crystal Structure and Properties of the Copper (II) Complex of a New Dioxocyclam Appended with Two 8-Methylquinolines as Additional Donor Pendants" Inorg. Chim. Acta.249. 125-130 (1996)
• [Publications] T. Koike et. al: "Intrinsic Properties of Zinc (II) Ion Pertinent to Zinc Enzymes" Advance in Inorganic Chemistry', Ed. by A. G. Sykes, Academic Press,. 44. 229-261 (1996)
• [Publications] T. Watanabe: "A Novel Biomimetic Zinc (II)-Fluorophore, Dansylamin oethyl-Pendant Macrocyclic Tetraamine 1, 4, 7, 10-Tetraazacyclododecane (Cyclen)" J. Am. Chem. Soc.118. 12696-12730 (1996)
• [Publications] S. Aoki et. al: "A This (Zinc (II) -1. 4. 7. 10-Cecraazacyclododecane) Complex as a New Receptor for Phosphate Dianions in Aqueous Solution" J. Am. Chem. Soc.119(in press). (1997)
• [Publications] M. Shionoya et. al: "Zinc Complexes as Targeting Agents for Nucleic Acids" "Metal Ions in Biological Systems" ed. by A. Sigel and H. Sigel, Marcel Dekker, Inc,. 33. 29-52 (1996)
• [Publications] M. Shionoya et. al: "Macrocyclic Metal Complexes for Selective Recognition of Nucleic Acid Bases and Manipulation of Gene Expression." Pure & Appl. chem.(in press).
• [Publications] Y. Sugiura et. al: "Distinct Phosphate Backbone Contacts revealed by Some Mutant Peptides of Zinc Finger Protein Sp1 : Effect of Protein-Induced Bending on DNA Recognition" Biochemistry. 35. 8761-8768 (1996)