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Molecular analyses of telomere regulation mechanism by telomere binding protein Pot1

Research Project

Project/Area Number 16390083
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field General medical chemistry
Research InstitutionTokyo University of Science

Principal Investigator

TORIGOE Hidetaka  Tokyo University of Science, Faculty of Science, Department of Applied Chemistry, Associate Professor, 理学部, 助教授 (80227678)

Co-Investigator(Kenkyū-buntansha) OGATA Kazuhiro  Yokohama City University, School of Medicine, Department of Biochemistry, Professor, 医学部, 教授 (90260330)
Project Period (FY) 2004 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 2006: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥6,900,000 (Direct Cost: ¥6,900,000)
KeywordsTelomere binding protein / Single-stranded telomeric DNA / Telomerase / Protein-DNA interaction / Protein-protein interaction / 4本鎖DNA構造 / 未変性ゲル電気泳動 / CDスペクトル / 蛍光スペクトル / ゲルシフト法 / two-hybrid法 / Pull-down法 / 免疫沈降法 / 欠失変異株 / Pot1
Research Abstract

We previously showed that the N-terminal region of Potl ranging from amino acids 1 to 182 is a single-stranded telomeric DNA-binding domain (Pot1DBD). We already established the E. coli overexpression system and the purification scheme of Pot1DBD. In this study, we have analyzed the three-dimensional structure of the purified Pot1DBD, and found that Pot1DBD was composed of three a-helices and eight β-strands and it belonged to OB (oligonucleotide/ oligosaccharide binding)-fold family.
Next, we analyzed the interaction between a series of mutated single-stranded telomeric DNA with various lengths and base sequences and the wild-type Pot1DBD by gel shift assay. A six base single-stranded DNA, d(GGTTAC), was the minimum telomeric DNA sequence for the specific binding of Pot1DBD. We also analyzed the interaction between a series of mutant Pot1DBD proteins and the telomeric DNA, d(GGTTAC), by gel shift assay. The binding ability of S58A, K90A and D125A was not significantly changed, but the … More binding ability of D64A, Q120L and K124A was significantly reduced in comparison with that of the wild-type Pot1DBD. The contribution to the complex formation was quite different in magnitude among the amino acids judged to contact with d(GGTTAC) by the three-dimensional structure of the complex.
Native gel electrophoresis and CD spectroscopy revealed that a fission yeast telomeric DNA, 4G4: d(G4TTAC)4, formed an antiparallel tetraplex DNA structure in the presence of 150 mM NaCl. The CD spectral change upon the addition of Pot1DBD to the antiparallel tetraplex of 4G4 indicated that Pot1DBD was able to reduce the amount of the antiparallel tetraplex DNA. Also, the fluorescence spectral change upon the addition of Pot1DBD to the antiparallel tetraplex of 4G4 attached by fluorophore and quencher to the 5' and 3' termini, respectively, showed that Pot1DBD was able to unfold the antiparallel tetraplex DNA. The interaction with a series of mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding.
On the other hand, we explored the proteins to interact with the C-terminal region of Potl (Potl C) by yeast two-hybrid screening using S. pombe cDNA library. One of the isolated candidates was Garl, a component of small nucleolar ribonucleoprotein particles (snoRNPs). The direct in vitro interaction between Potl C and Garl was confirmed by pull-down and immunoprecipitation assays. The gar/-knockout mutant constructed to analyze the effect of Garl on the telomere length was lethal. In the knockout mutant, Potl was not able to recruit telomerase due to the absence of Garl, which may cause severe telomere shortening and lethality. These results hypothesize that Potl can associate with telomerase indirectly through the interaction with Garl. Less

Report

(4 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • Research Products

    (36 results)

All 2007 2006 2005 2004

All Journal Article (33 results) Book (3 results)

  • [Journal Article] Tetraplex Structure of Fission Yeast Telomeric DNA and Unfolding of the Tetraplex on the Interaction with Telomeric DNA Binding Protein Pot12007

    • Author(s)
      Torigoe, H., Furukawa, A.
    • Journal Title

      J. Biochem. 141

      Pages: 57-68

    • NAID

      10020101926

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Mutational Analyses of a Single-stranded Telomeric DNA Binding Domain of Fission Yeast Pot1 : Conflict with X-ray Crystallographic Structure2007

    • Author(s)
      Torigoe, H., Dohmae, N., Hanaoka, F., Furukawa, A.
    • Journal Title

      Biosci. Biotech. Biochem. 71

      Pages: 481-490

    • NAID

      10027511703

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Tetraplex Structure of Fission Yeast Telomeric DNA and Unfolding of the Tetraplex on the Interaction with Telomeric DNA Binding Protein Potl2007

    • Author(s)
      Torigoe, H, Furukawa, A.
    • Journal Title

      J. Biochem. 141

      Pages: 57-68

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Mutational Analyses of a Single-stranded Telomeric DNA Binding Domain of Fission Yeast Potl : Conflict with X-ray Crystallographic Structure2007

    • Author(s)
      Torigoe, H, Dohmae, N, Hanaoka, F, Furukawa, A.
    • Journal Title

      Biosci. Biotech. Biochem., 71

      Pages: 481-490

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Detection of C : C Mismatch Base Pair by Fluorescence Spectral Change upon Addition of Silver (I) Cation : Toward the Efficient Analyses of Single Nucleotide Polymorphism2006

    • Author(s)
      Torigoe, H., Kozasa, T., Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 89-90

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection of T : T Mismatch Base Pair by Fluorescence Spectral Change upon Addition of Mercury (II) Cation : Toward the Efficient Analyses of Single Nucleotide Polymorphism2006

    • Author(s)
      Torigoe, H., Kozasa, T., Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 223-224

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Thermodynamic Analyses of the Specific Interaction between Two C : C Mismatch Base Pairs and Silver (I) Cations2006

    • Author(s)
      Torigoe, H., Miyakawa, Y., Nagasawa, N., Kozasa, T., Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 225-226

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection of C : C Mismatch Base Pair by Fluorescence Spectral Change upon Addition of Silver (I) Cation : toward the Efficient Analyses of Single Nucleotide Polymorphism2006

    • Author(s)
      Torigoe, H, Kozasa, T, Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 89-90

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Detection of T : T Mismatch Base Pair by Fluorescence Spectral Change upon Addition of Mercury (II) Cation : Toward the Efficient Analyses of Single Nucleotide Polymorphism2006

    • Author(s)
      Torigoe, H, Kozasa, T, Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 223-224

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Thermodynamic analyses of the Specific Interaction between Two C : C Mismatch Base Pairs and Silver (I) Cations2006

    • Author(s)
      Torigoe, H, Miyakawa, Y, Nagasawa, N, Kozasa, T., Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 50

      Pages: 225-226

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Combination of Poly(L-lysine)-graft-dextran Copolymer and 2' -0,4' -C- methylene Bridged Nucleic Acid (2',4' -BNA) Modification Synergistically Stabilizes Pyrimidine Motif Triplex at Neutral pH2005

    • Author(s)
      Torigoe, H., Katayama, T., Obika, S., Maruyama, A., Imanishi, T.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 635-638

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Novel Strategy for Single Nucleotide Polymorphism (SNP) Genotyping by Heteroduplex Analysis : Specific Stabilization of TT Mismatch Base Pair by Mercury (II) Cation and CC Mismatch Base Pair by Silver (I) Cation2005

    • Author(s)
      Torigoe, H., Kawahashi, K., Takamori, A., Ono, A.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 915-917

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Promotion of Pyrimidine Motif Triplex Formation by Morpholino Modification of Triplex-forming Oligonucleotide : Kinetic and Thermodynamic Studies2005

    • Author(s)
      Torigoe, H., Kawahashi, K., Tamura, Y.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 1019-1021

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Tetraplex Structure of Fission Yeast Telomeric DNA and Its Unfolding by the Interaction with Telomeric DNA Binding Protein Pot12005

    • Author(s)
      Furukawa, A., Torigoe.H.
    • Journal Title

      Nucleic Acids Symp. Ser. 49

      Pages: 63-64

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Synergistic Stabilization of Nucleic Acid Assembly by Oligo-N3'-P5' Phosphoramidate Modification and Additions of Comb-type Cationic Copolymers2005

    • Author(s)
      Torigoe, H., Maruyama, A.
    • Journal Title

      J. Am. Chem. Soc. 127

      Pages: 1705-1710

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Combination of Poly (L-lysine)-graft-dextran Copolymer and 2'-0,4'-C-methylene Bridged Nucleic Acid (2',4'-BNA) Modification Synergistically Stabilizes Pyrimidine Motif Triplex at Neutral pH2005

    • Author(s)
      Torigoe, H., Katayama, T., Obika, S., Maruyama, A., Imanishi, T.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 635-638

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Tetraplex Structure of Fission Yeast Telomeric DNA and Its Unfolding by the Interaction with Telomeric DNA Binding Protein Pot12005

    • Author(s)
      Furukawa, A., Torigoe, H.
    • Journal Title

      Nucleic Acids Symp. Ser. 49

      Pages: 63-64

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Thermodynamic Analyses of the Specific Interaction between C : C Mismatch Base Pair and Silver (I) Cation2005

    • Author(s)
      Torigoe, H., Kozasa, T., Takamori, A., Ono, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 49

      Pages: 217-218

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Development of Triplex Formation- based Artificial Transcription Factor to Recognize Any Upstream Sequence of Target Genes2005

    • Author(s)
      Torigoe, H., Tsukamoto, Y.
    • Journal Title

      Nucleic Acids Symp. Ser. 49

      Pages: 321-322

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Computational Evaluation of the Specific Interaction between Cation and Mismatch Base Pair2005

    • Author(s)
      Sugiyama, H., Adachi, N., Kawauchi, S., Kozasa, T., Katayama, T., Torigoe, H., Ono, A., Tamura, Y.
    • Journal Title

      Nucleic Acids Symp. Ser. 49

      Pages: 215-216

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Combination of Poly(L-lysine)-graft-dextran Copolymer and 2'-O,4'-C-methylene Bridged Nucleic Acid (2',4'-BNA) Modification Synergistically Stabilizes Pyrimidine Motif Triplex at Neutral pH2005

    • Author(s)
      Torigoe, H., Katayama, T., Obika, S.Maruyama, A., Imanishi, T.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 635-638

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Novel Strategy for Single Nucleotide Polymorphism (SNP) Genotyping by Heteroduplex Analysis : Specific Stabilization of TT Mismatch Base Pair by Mercury (II) Cation and CC Mismatch Base Pair by Silver (I) Cation2005

    • Author(s)
      Torigoe, H., Kawahashi, K., Takamori, A., Ono, A.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 915-917

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Promotion of Pyrimidine Motif Triplex Formation by Morpholino Modification of Triplex-forming Oligonucleotide : Kinetic and Thermodynamic Studies2005

    • Author(s)
      Torigoe, H., Kawahashi, K., Tamura, Y.
    • Journal Title

      Nucleosides, Nucleotides & Nucleic Acids 24

      Pages: 1019-1021

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Tetraplex Structure of Fission Yeast Telomeric DNA and Its Unfolding by the Interaction with Telomeric DNA Binding Protein Pot12005

    • Author(s)
      Furukawa, A., Torigoe, H.
    • Journal Title

      Nucleic Acids Symp.Ser. 49

      Pages: 63-64

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Thermodynamic Analyses of the Specific Interaction between C:C Mismatch Base Pair and Silver (I) Cation : Toward the Efficient Detection of Single Nucleotide Polymorphism2005

    • Author(s)
      Torigoe, H., Kozasa, T., Takamori, A., Ono, A.
    • Journal Title

      Nucleic Acids Symp.Ser. 49

      Pages: 217-218

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Development of Triplex Formation-based Artificial Transcription Factor to Recognize Any Upstream Sequence of Target Genes2005

    • Author(s)
      Torigoe, H., Tsukamoto, Y.
    • Journal Title

      Nucleic Acids Symp.Ser. 49

      Pages: 321-322

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Synergistic Stabilization of Nucleic Acid Assembly by Oligo-N3'-P5' Phosphoramidate Modification and Additions of Comb-type Cationic Copolymers2005

    • Author(s)
      Torigoe, H., Maruyama, A.
    • Journal Title

      J.Am.Chem.Soc. 127

      Pages: 1705-1710

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Thermodynamic Analyses of the Specific Interaction between T : T Mismatch Base Pair and Mercury (II) Cation2004

    • Author(s)
      Torigoe, H., Ono, A., Kawahashi, K.
    • Journal Title

      Nucleic Acids Symp. Ser. 48

      Pages: 275-276

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Silver (I) Cation Specifically Stabilizes Heteroduplex with C : C Mismatch Base Pair2004

    • Author(s)
      Torigoe, H., Ono, A., Takamori, A.
    • Journal Title

      Nucleic Acids Symp. Ser. 48

      Pages: 101-102

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Synergistic Stabilization of Triplex by Combination of Comb-type Cationic Copolymer and 2',4'-BNA2004

    • Author(s)
      Katayama, T., Maruyama, A., Obika, S., Imanishi, T., Torigoe, H.
    • Journal Title

      Nucleic Acids Symp. Ser. 48

      Pages: 139-140

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Thermodynamic Analyses of the Specific Interaction between T:T Mismatch Base Pair and Mercury (II) Cation2004

    • Author(s)
      Torigoe, H., Ono, A., Kawahashi K.
    • Journal Title

      Nucleic Acids Symp.Ser. 48

      Pages: 275-276

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Silver (I) Cation Specifically Stabilizes Heteroduplex with C:C Mismatch Base Pair2004

    • Author(s)
      Torigoe, H., Ono, A., Takamori, A.
    • Journal Title

      Nucleic Acids Symp.Ser. 48

      Pages: 101-102

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Synergistic Stabilization of Triplex by Combination of Comb-type Cationic Copolymer and 2'4'-BNA2004

    • Author(s)
      Katayama, T., Maruyama, A., Obika, S., Imanishi, T., Torigoe, H.
    • Journal Title

      Nucleic Acids Symp.Ser. 48

      Pages: 139-140

    • Related Report
      2004 Annual Research Report
  • [Book] Novel Strategy for Efficient Detection of Single Nucleotide Polymorphism (SNP) : Specific Binding of Mercury (II) Cation to T : T Mismatch Base Pair and That of Silver (I) Cation to C : C Mismatch Base Pair" in "Moleculomics and Thereafter"2006

    • Author(s)
      Torigoe, H., Ono, A.
    • Total Pages
      21
    • Publisher
      Research Signpost, Ltd.
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Book] Moleculomics and Thereafter2006

    • Author(s)
      Torigoe, H, Ono, A.
    • Total Pages
      157
    • Publisher
      Research Signpost, Ltd.
    • Related Report
      2006 Annual Research Report
  • [Book] "Novel Strategy for Efficient Detection of Single Nucleotide Polymorphism (SNP) : Specific Binding of Mercury (II) Cation to T:T Mismatch Base Pair and That of Silver (I) Cation to C :C Mismatch Base Pair" "Moleculomics and Thereafter"2006

    • Author(s)
      Torigoe, H., Ono, A.
    • Total Pages
      21
    • Publisher
      Research Signpost, Ltd.
    • Related Report
      2005 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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