| 研究課題/領域番号 |
21K20645
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| 研究種目 |
研究活動スタート支援
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| 配分区分 | 基金 |
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| 審査区分 |
0701:分子レベルから細胞レベルの生物学およびその関連分野
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
AYALA・HERNANDEZ Rafael 沖縄科学技術大学院大学, 生体分子電子顕微鏡解析ユニット, ポストドクトラルスカラー (00912601)
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| 研究期間 (年度) |
2021-08-30 – 2025-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
3,120千円 (直接経費: 2,400千円、間接経費: 720千円)
2022年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
2021年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
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| キーワード | DNA complex / cryo-EM / structural biology / telomeres / cancer / aging / molecular biology / cryoEM / chromatin / Chromatin / Cryo-EM / Telomeres / DNA damage |
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| 研究開始時の研究の概要 |
First, recombinant human shelterin complexes will be expressed in a baculovirus-insect cell expression system. The complexes will be purified and their structure will be solved by means of cryo-EM. The structure will be solved both in the absence and presence of a telomeric DNA substrate. The resulting high-resolution structures will reveal the architecture of the shelterin complex and the mechanism of recognition and binding to telomeric DNA.
Later, higher order structures of shelterin will be investigated by using longer DNA substrates, such as a mimic of the telomeric t-loop found in cells.
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| 研究実績の概要 |
Shelterin complex without a DNA substrates has been confirmed to be too flexible for cryo-EM studies by single particle, even after crosslinking. We have now prepared DNA substrates mimicking telomeric DNA, and have changed our focus to analyzing the shelterin-DNA complex.
Additionally, the learnt methods and techniques have also been applied to study the DT57C bacteriophage, which has resulted in the publication of a research paper at Nature Communications: "Nearly complete structure of bacteriophage DT57C reveals architecture of head-to-tail interface and lateral tail fibers". The paper has presented for the first time a molecular model of an entire siphophage comprising all the core structural components.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
While difficulties in analyzing the structure of the human shelterin complex have been found due to its inherent flexibility, we have obtained valuable information that has allowed us to determine that focus should be changed to analyzing the complex with a DNA substrate.
Additionally, the techniques and methodologies learnt along the process have also been applied to an entire bacteriophage (DT57C), leading to a high-impact publication.
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| 今後の研究の推進方策 |
The complex of shelterin with a DNA substrate mimicking will be determined by cryo-EM.
Additionally, application of the same techniques to other bacteriophages of interest will be tested, given the recent success with DT57C.
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