| Project/Area Number |
21K20645
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| Research Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Multi-year Fund |
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| Review Section |
0701:Biology at molecular to cellular levels, and related fields
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
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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| Project Period (FY) |
2021-08-30 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2021: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | DNA complex / cryo-EM / structural biology / telomeres / cancer / aging / molecular biology / cryoEM / chromatin / Chromatin / Cryo-EM / Telomeres / DNA damage |
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| Outline of Research at the Start |
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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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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