Cryo-EM analysis of the 3D-structure and mechanism of okazakisome
| Project/Area Number |
18K06089
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 43020:Structural biochemistry-related
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| Research Institution | Kyushu University |
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Principal Investigator |
Mayanagi Kouta 九州大学, 生体防御医学研究所, 助教 (50418571)
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| Co-Investigator(Kenkyū-buntansha) |
石野 良純 九州大学, 農学研究院, 教授 (30346837)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | 単粒子解析 / 電子顕微鏡 / 超分子複合体 / 立体構造解析 / DNA複製 / オカザキソーム / DNAクランプ / 生物物理 / クライオ電子顕微鏡 |
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| Outline of Final Research Achievements |
We investigated the 3D-structure of FEN-PCNA-DNA, which plays a key role in okazakisome, the lagging strand replication machine. We also successfully obtained the intermediate state, FEN-Lig-PCNA-DNA complex, in which DNA was being handed over from FEN to Lig.
PolD is the genome DNA replicating enzyme and belongs to the family D polymerase. So far, no structure of proteins belonging to this family has been obtained.
We studied the structure of PolD and its complex with PCNA and DNA, using cryo-EM single particle analysis. The complex exhibited two distinct forms, which were assigned to DNA polymerizing mode and DNA editing mode. Also, we analyzed the structure of nucleosome complexed with AID segment of FACT. The obtained structure revealed the mechanism of the initial step of nucleosome remodeling by FACT.
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| Academic Significance and Societal Importance of the Research Achievements |
オカザキソームによるラギング鎖の複製は主にポリメラーゼ、FEN、Ligによって３ステップで進行する。これまで唯一構造が未知であったFEN-PCNA-DNAの構造を明らかした。また2個の因子間でDNAが手渡しされることは、塩基除去修復の系で仮説として提唱されていたが、DNA複製の系で実際にその瞬間を捉えたのは初めてである。PolDはPCR等で利用されるPolBと同様に古細菌のDNA複製酵素であり、非常に高い酵素活性をもつ。しかしながら７つのファミリーのうちPolDのファミリーのみこれまで構造的知見が乏しかった。本解析によってPolDの生命科学ツールとしての応用も期待される。
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Report
(4 results)
Research Products
(61 results)
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[Presentation] Integrative Analysis Combining AFM, NMR, and EM Revealed Two-State Conformational Exchange of a Single-subunit Oligosaccharyltransferase from Archaeoglobus fulgidus.2019
Author(s)
Yuki Kawasaki, Hirotaka Ariyama, Daisuke Fujinami, Hajime Motomura, Ashutosh Srivastava, Florence Tama, Daisuke Noshiro, Toshio Ando, Kouta Mayanagi, Daisuke Kohda
Organizer
15th International Congress on Thermophiles
Related Report
Int'l Joint Research
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[Presentation] Replisome structure and its functions in Archaea2018
Author(s)
Yoshizumi Ishino, Keisuke Oki, Mariko Nagata, Kouta Mayanagi, Tsuyoshi Shirai, Takuji Oyama, Kosuke Morikawa, Takeshi Yamagami, and Sonoko Ishino
Organizer
OKAZAKI Fragment Memorial Symposium: Celebrating the 50th anniversary of the discontinuous DNA replication model
Related Report
Int'l Joint Research / Invited
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