Elucidation of the mecahsim of thermostabilization of thermostable reverse transcriptase

• Project/Area Number: 15K07388
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Kyoto University
• Principal Investigator: YASUKAWA KIYOSHI 京都大学, 農学研究科, 教授 (30397559)
• Co-Investigator(Kenkyū-buntansha): 兒島 憲二 京都大学, 農学研究科, 助教 (40542759)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: リボヌクレアーゼH / RNase H / RNase H2 / 逆転写酵素 / GPMA method / CRISPR/Cas9システム / 変異原性 / MMLV RT / ゲノムプロフィリング法 / 耐熱化機構

Outline of Final Research Achievements

Ribonuclease H (RNase H) is an enzyme that specifically degrades RNA from RNA/DNA hybrids. RNase H has been classified into two major families, type 1 and type 2. Retroviral reverse transcriptase (RT) has DNA polymerase and RNase H activities. In this study, we aimed to elucidate the RNase H and engaged in the following four subjects. (i) The RNase H domain of MMLV RT was prepared and characterized. (ii) The effects of neutral salts and pH on the activity and stability of human RNase H2 were analyzed. (iii) The GPMA method using mouse NIH3T3 cell line instead of E. coli was established. (iv) The RNase H2 knock-out NIH3T3 cell line was established and characterized.

Research Products

• [Int'l Joint Research] National Institutes of Health(米国)
• [Int'l Joint Research] バンドン工科大学(インドネシア)
• [Int'l Joint Research] National Institutes of Health(米国)
• [Journal Article] Effects of neutral salts and pH on the activity and stability of human RNase H2 (2017)
  • Author(s): Baba, M., Kojima, K., Nakase, R., Imai, S., Yamasaki, T., Takita, T., Crouch, R.J., Yasukawa, K.
  • Journal Title: The Journal of Biochemistry Volume: 印刷中 Issue: 3 Pages: 211-219
  • DOI: 10.1093/jb/mvx021
  • NAID: 40021326122
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] DNA-based mutation assay (GPMA) can be of ppb-order sensitivity for both bacterial and mammalian testers (2017)
  • Author(s): Kumari, P., Gautam, S.G., Baba, M., Tsukiashi, M., Matsuoka, K., Yasukawa, K., Nishigaki, K.
  • Journal Title: The Journal of Biochemistry Volume: 162 Issue: 6 Pages: 395-401
  • DOI: 10.1093/jb/mvx043
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system (2017)
  • Author(s): Katano, Y., Li, T., Baba, M., Nakamura, M., Ito, M., Kojima, K., Takita, T., Yasukawa, K.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 81 Issue: 12 Pages: 2339-2345
  • DOI: 10.1080/09168451.2017.1394790
  • Peer Reviewed
• [Journal Article] Further increase in thermostability of Moloney murine leukemia virus reverse transcriptase by mutational combination (2017)
  • Author(s): Baba, M., Kakue, R., Leucht, C., Rasor, P., Walch, H., Ladiges, D., Bell, C., Kojima, K., Takita, T., Yasukawa, K.
  • Journal Title: Protein Eng. Des. Sel. Volume: 30 Issue: 8 Pages: 551-557
  • DOI: 10.1093/protein/gzx046
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase (2015)
  • Author(s): Nishimura, K., Yokokawa, K., Hisayoshi, T., Fukatsu, K., Kuze, I., Konishi, A., Mikami, B., Kojima, K., and Yasukawa, K.
  • Journal Title: Protein Expression and Purification Volume: 113 Pages: 44-50
  • DOI: 10.1016/j.pep.2015.04.012
  • NAID: 120005668067
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Site saturation変異と無細胞タンパク質合成系を用いた耐熱化MMLV 逆転写酵素スクリーニング系の構築 (2017)
  • Author(s): 李瞳陽、片野裕太、中村実世、馬場美聡、伊東昌章、滝田禎亮、保川清
  • Organizer: 日本農芸化学会2017年度大会
  • Place of Presentation: 京都女子大学
  • Year and Date: 2017-03-17
• [Presentation] ヒトRNase H2の酵素活性と安定性に与える中性塩とpHの影響 (2017)
  • Author(s): 馬場美聡，兒島憲二，中瀬理保子，今井翔太，山崎朋美，滝田禎亮，Robert J. Crouch，保川清
  • Organizer: 第64回日本生化学会近畿支部例会
• [Presentation] 哺乳動物細胞を用いたGPMA（genome profiling-based mutation assay）法の検討 (2017)
  • Author(s): 馬場美聡、Parmila Kumari、Sunita Ghimire Gautam、月足元希、松岡浩司、西垣功一、保川清
  • Organizer: 第46回日本環境変異原学会大会
• [Presentation] ヒトリボヌクレアーゼ H2の基質特異性の改変 (2017)
  • Author(s): 兒島憲二，馬場美聡，杉浦拓也，西村拓人，滝田禎亮，Robert J. Crouch，保川清
  • Organizer: 日本農芸化学会平成29年度関西支部大会（第500回講演会）
• [Presentation] 哺乳動物細胞を用いたゲノムプロフィリング法による変異原アッセイ法の検討 (2017)
  • Author(s): 馬場美聡、Parmila Kumari、Sunita Ghimire Gautam、月足元希、松岡浩司、西垣功一、保川清
  • Organizer: 日本農芸化学会平成29年度関西支部大会（第500回講演会）
• [Presentation] ヒトリボヌクレアーゼ H2の基質特異性の改変 (2017)
  • Author(s): 兒島憲二，馬場美聡，杉浦拓也，西村拓人，滝田禎亮，Robert J. Crouch，保川清
  • Organizer: 2017年度生命科学系合同年次大会
• [Presentation] 哺乳動物細胞を用いたGPMA (genome profiling-based mutation assay)法の検討 (2017)
  • Author(s): 馬場美聡、Parmila Kumari、Sunita Ghimire Gautam、月足元希、松岡浩司、西垣功一、保川清
  • Organizer: 2017年度生命科学系合同年次大会
• [Presentation] マウス線維芽細胞株NIH3T3 RNase H2遺伝子欠損株の作製 (2017)
  • Author(s): 月足元希、馬場美聡、兒島憲二、滝田禎亮、保川清
  • Organizer: 2017年度生命科学系合同年次大会
• [Presentation] 部位特異的変異導入によるヒトRNase H2の基質特異性の改変 (2017)
  • Author(s): 兒島憲二，馬場美聡，西村拓人，杉浦拓也，滝田禎亮，上原了，Robert J. Crouch，保川清
  • Organizer: ２０１８年度日本農芸化学会大会
• [Presentation] 変異の組み合わせによるモロニ―マウス白血病ウイルス逆転写酵素のさらなる耐熱化 (2017)
  • Author(s): 馬場美聡, 角江亮太, Christoph Leucht, Peter Rasor, Heiko Walch, Daniel Ladiges, Christian Bell, 兒島憲二, 滝田禎亮, 保川清
  • Organizer: ２０１８年度日本農芸化学会大会
• [Presentation] Effects of neutral salts on catalytic activity and stability of human RNase H2 (2016)
  • Author(s): Kojima, K., Baba, M., Kojima, K., Nakase, R., Imai, S., Yamasaki, Crouch, R.J., T., Takita, T., Yasukawa, K.
  • Organizer: RNaseH2016
  • Place of Presentation: 京都大学楽友会館
  • Year and Date: 2016-09-06
  • Int'l Joint Research
• [Presentation] ヒトRNase H2の活性と安定性に対する塩の影響 (2016)
  • Author(s): 兒島憲二、馬場美聡、中瀬理保子、今井翔太、山﨑朋美、Robert J. Crouch、滝田禎亮、保川清
  • Organizer: 日本農芸化学会関西支部第495回講演会
  • Place of Presentation: 大阪府立大学
• [Presentation] Nase H2の酵素化学的性質の解析 (2016)
  • Author(s): 馬場美聡、兒島憲二、中瀬理保子、今井翔太、山崎朋美、Robert J. Crouch、滝田禎亮、保川清
  • Organizer: 日本農芸化学会関西支部第496回講演会
  • Place of Presentation: 滋賀県立大学
• [Presentation] Effects of neutral salts and pH on catalytic activity of human RNase H2 (2016)
  • Author(s): Baba, M., Kojima, K., Nakase, R., Imai, S., Yamasaki, T., Crouch, R.J., Takita, T.,Yasukawa, K.
  • Organizer: JSBBA Student Forum
  • Place of Presentation: 神戸大学
• [Presentation] MMLV 逆転写酵素のRNase Hドメインの性状解析 (2015)
  • Author(s): 横川貫太、西村耕作、深津浩佑、久世郁実、久好哲郎、小西篤、三上文三、兒島憲二、保川清
  • Organizer: 第６２回日本生化学会近畿支部例会
  • Place of Presentation: 立命館大学びわこ・くさつキャンパス
  • Year and Date: 2015-05-16
• [Book] エッセンシャルタンパク質工学 (2018)
  • Author(s): 老川典夫、大島敏久、保川清、三原久明、宮原郁子
  • Total Pages: 210
  • Publisher: 講談社
  • ISBN: 9784061538993
• [Remarks] 酵素化学研究室ホームページ 業績
  • URL: http://www.enzchem.kais.kyoto-u.ac.jp/gyouseki.html