2023 Fiscal Year Research-status Report
Architecture and mechanism of the shelterin complex
| Project/Area Number |
21K20645
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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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| Keywords | DNA complex / cryo-EM / structural biology |
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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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| Causes of Carryover |
The difficulties observed due to the flexibility of the shelterin complex made it difficult to complete structural characterization. A new way has been found, to make a complex with a DNA substrate, which is currently in the process of being tested during this fiscal year. The funds will be used to perform the cryo-EM analysis of the shelterin complex bound to a DNA substrate, to cover the costs of required consumables including grids for electron microscope and culture medium for the expression of shelterin in insect cells.
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