DNA上の極性を有したスライディングとカップルした塩基変換酵素反応の解析

• Project Area: Integrative understanding of biological processes mediated by transient macromolecular complexes; New technology for visualizing physiologically metastable states.
• Project/Area Number: 22121517
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: Kyoto University
• Principal Investigator: 片平 正人 京都大学, エネルギー理工学研究所, 教授 (70211844)
• Project Period (FY): 2010 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥10,920,000 (Direct Cost: ¥8,400,000、Indirect Cost: ¥2,520,000); Fiscal Year 2011: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2010: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
• Keywords: 生物物理 / タンパク質 / 分子認識 / 酵素反応 / APOBEC3G / HIV / デアミネーション / NMR / 抗HIV / リアルタイムモニタリング / DNA / 立体構造

Research Abstract

ヒトのAPOBEC3G(以下A3G)タンパク質は、HIVのDNAに作用して、シトシンをデアミネーションしてウラシルに変換する酵素である。これによりHIVのゲノム情報は破壊され、A3Gは抗HIV活性を有する。我々はNMRシグナルを用いる事で、A3Gによるデアミネーション反応をリアルタイムでモニタリングできる事を、これまでに報告してきた。
今回デアミネーション反応のホットスポットを2つ有する1本鎖DNA及び同ホットスポットを3つ有する2つ有する1本鎖DNAを調製した。この際、特定のシトシンに13C安定同位体標識を施した。この標識により、どのシトシンがデアミネーションを受けたのかを一義的に帰属・同定する事が可能となった。標識によって、これまでより長鎖のDNAにおけるデアミネーションも比較的容易に追跡できる事が実証された。
DNAにA3Gを作用させ、各ホットスポットおけるデアミネーションの進行を、申請者等が確立したリアルタイムモニタリングの手法で追跡した。その結果我々の予想通り5'端近くのホットスポットの方が、3'端近くのホットスポットより、デアミネーションを受ける頻度が高い事が実証された。またホットスポット間に2本鎖を形成させ、想定されているA3Gのスライディングを阻害した系では、ホットスポット間におけるデアミネーション頻度の差が消失する事が確認された。
デアミネーション効率に関して各ホットスポットにおいて得られた実験データを、カイネティックモデルを構築して再現する事を試みた。その結果、A3GはDNA鎖上をスライディングすると考えられる事、そしてスライディングの方向によってホットスポットにおける触媒活性が異なると考えられる事が示唆された。HIVのゲノムDNAの広い範囲に渡ってA3Gが効率的に変異を導入する上で、この事は有利に働いていると考えられる。

Research Products

• [Journal Article] Structure of Musashil in a complex with target RNA : The role of aromatic stacking interactions (2012)
  • Author(s): Ohyama, et al
  • Journal Title: Nucleic Acids Res. Volume: (in press)
  • Peer Reviewed
• [Journal Article] Intelligent ribozyme that switches its activity in response to K+ through Quadruplex formation (2012)
  • Author(s): Nagata, et al
  • Journal Title: FEBS J. Volume: 279 Pages: 1456-1463
  • Peer Reviewed
• [Journal Article] Structural aspects for the recognition of ATP by ribonucleopeptide receptors (2011)
  • Author(s): Nakano, et al
  • Journal Title: J.Am.Chem.Soc. Volume: 133 Pages: 4567-4579
  • Peer Reviewed
• [Journal Article] Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G (2011)
  • Author(s): Sugiyama, et al
  • Journal Title: J.Biol.Chem. Volume: 286 Pages: 10051-10057
  • Peer Reviewed
• [Journal Article] Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase (2011)
  • Author(s): Kondo, K
  • Journal Title: Internat.J.of Biolog.Macromol. Volume: VOL.48 Pages: 256-262
  • Peer Reviewed
• [Journal Article] Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G (2011)
  • Author(s): Sugiyama, R.
  • Journal Title: J.Biol.Chem Volume: (印刷中 掲載確定)
  • Peer Reviewed
• [Journal Article] Structural aspects for the recognition of ATP by ribonucleopeptide receptors (2011)
  • Author(s): Nakano, S.
  • Journal Title: J.Am.Chem.Soc. Volume: VOL.133 Pages: 4567-4579
  • Peer Reviewed
• [Journal Article] Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodnii (2010)
  • Author(s): Takeda, M.
  • Journal Title: Int.J.Biol.Macromol Volume: VOL.46 Pages: 206-211
  • Peer Reviewed
• [Journal Article] Solution structures of the trihelix DNA-binding domains of the wild-type and a phosphomimetic mutant of Arabidopsis GT-1, implying a mechanism for an increase in DNA-binding affinity through phosphorylation (2010)
  • Author(s): Nagata, T.
  • Journal Title: Proteins Volume: VOL.78 Pages: 3033-3047
  • Peer Reviewed
• [Presentation] Sliding-direction-dependent activity of anti-HIV enzyme, and structural basis of anti-prion activity of RNA aptamer (2012)
  • Author(s): Furukawa, et al
  • Organizer: Korea-Japan Bilateral NMR Symposium
  • Place of Presentation: 北海道大学(招待講演)
  • Year and Date: 2012-03-16
• [Presentation] Base-deamination rate dependent on the direction of sliding of an enzyme along DNA as revealed by numerical aialysls of NMR data, and wood biomass studied by solution NMR (2011)
  • Author(s): Nishimura, et al
  • Organizer: The International Symposium on Nuclear Magnetic Resonance 2011
  • Place of Presentation: 大桟橋ホール(神奈川県)
  • Year and Date: 2011-11-17
• [Presentation] Real-time monitoring of the cytidine deamination along single-stranded DNA by an anti-HIV factor, APOBEC3G (2011)
  • Author(s): Furukawa, et al
  • Organizer: The International Symposium on Nuclear Magnetic Resonance 2011
  • Place of Presentation: 大桟橋ホール(神奈川県)
  • Year and Date: 2011-11-16
• [Presentation] r (GGA) 4 chimeric ribozyme switches its activity in response to K+ via quadruplex formation (2011)
  • Author(s): Nagata, et al
  • Organizer: The 38th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: 北海道大学
  • Year and Date: 2011-11-11
• [Presentation] Structural basis of the high affinity of the RNA aptamer against prion protein (2011)
  • Author(s): Mashima, et al
  • Organizer: The 16th annual meeting of the RNA society
  • Place of Presentation: プリンスホテル(京都府)
  • Year and Date: 2011-06-15
• [Presentation] Linkage analysis of lignin for lignocellulosic biorefinery (2010)
  • Author(s): Yoshioka, K.
  • Organizer: The International Chemical Congress of Pacific Basin Societies
  • Place of Presentation: ハワイコンベンションセンター(米国)
  • Year and Date: 2010-12-17
• [Presentation] Unique quadruplex structure and interaction of r(GGAGGAGGAGGA) with protein (2010)
  • Author(s): Mashima, T.
  • Organizer: The International Chemical Congress of Pacific Basin Societies
  • Place of Presentation: ハワイコンベンションセンター(米国)
  • Year and Date: 2010-12-16
• [Presentation] Structure and interaction of an RNA aptamer with potential therapeutic use (2010)
  • Author(s): Mashima, T.
  • Organizer: The International Chemical Congress of Pacific Basin Societies
  • Place of Presentation: ハワイコンベンションセンター(米国)
  • Year and Date: 2010-12-15
• [Presentation] The real-time monitoring of the enzymatic reaction of an anti-HIV factor APOBEC3G (2010)
  • Author(s): Furukawa, A.
  • Organizer: Korea-Japan Bilateral NMR Symposium
  • Place of Presentation: ソウル大学(韓国)
  • Year and Date: 2010-11-26
• [Presentation] A mechanism for a increase in DNA-binding affinity of the trihelix DNA-binding domain via its phosphrylation (2010)
  • Author(s): Nagata, T.
  • Organizer: The 37^<th> International Symposium on Nucleic Acids Chemistry 2010
  • Place of Presentation: はまぎんホール(横浜市)
  • Year and Date: 2010-11-11
• [Presentation] Structural aspects of the fluorescence intensity changes of the ATP-binding ribonucleopeptide sensor (2010)
  • Author(s): Nakano, S.
  • Organizer: The 37^<th> International Symposium on Nucleic Acids Chemistry 2010
  • Place of Presentation: はまぎんホール(横浜市)
  • Year and Date: 2010-11-11
• [Presentation] Structure and interaction of RNA aptamer against prion protein in complex with the partial binding peptide (2010)
  • Author(s): Mashima, T.
  • Organizer: The 37^<th> International Symposium on Nucleic Acids Chemistry 2010
  • Place of Presentation: はまぎんホール(横浜市)
  • Year and Date: 2010-11-10
• [Presentation] Real-time monitoring of the enzymatic reaction of APOBEC3G possessing anti-HIV activity, and the structure and interaction of the RNA aptamer against prion protein (2010)
  • Author(s): Furukawa, A.
  • Organizer: The 10^<th> KIAS Conference on Protein Structure and Function
  • Place of Presentation: International Conference Hall(韓国)
  • Year and Date: 2010-10-01
• [Presentation] The real-time monitoring of the enzymatic reaction of an anti-HIV factor APOBEC3G (2010)
  • Author(s): Furukawa, A.
  • Organizer: XXIV International Conference on Magnetic Resonance in Biological Systems
  • Place of Presentation: Cairns Convention Centre Cairns,(オーストラリア)
  • Year and Date: 2010-08-24
• [Presentation] Real-time monitoring of the enzymatic reaction of APOBEC3G possessing anti-HIV activity, and the structure and interaction of the RNA aptamer against prion protein (2010)
  • Author(s): Mashima, T.
  • Organizer: XXIV International Conference on Magnetic Resonance in Biological Systems
  • Place of Presentation: Cairns Convention Centre Cairns(オーストラリア)
  • Year and Date: 2010-08-23
• [Presentation] The analysis of the mechanism of an enzyme reaction toward the development of new energy systems (2010)
  • Author(s): Iwaoka, R.
  • Organizer: The 2^<rd> International Symposium of Kyoto University Global COE Program-Zero Carbon Energy Kyoto 2010
  • Place of Presentation: 京都大学おうばくプラザ(宇治市)
  • Year and Date: 2010-08-19
• [Presentation] Real-time monitoring of the enzymatic reaction of APOBEC3G possessing anti-HIV activity, and the structure of the RNA aptamer against prion protein (2010)
  • Author(s): Furukawa, A.
  • Organizer: International Symposium of Korean Society of Biochemistry and Molecular Biology -Structural Biology and Drug Discovery-
  • Place of Presentation: ソウル大学(韓国)
  • Year and Date: 2010-05-18
• [Book] CSJカレントレビュー"核酸化学のニュートレンド-DNA・RNAの新たな可能性を開く"(日本化学会編) (2011)
  • Author(s): 片平正人
  • Total Pages: 5
  • Publisher: 化学同人
• [Remarks]
  • URL: http://www.iae.kyoto-u.ac.jp/bio/