2021-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/654578"端粒是真核染色體的末端結構。它們是維持染色體完整性所必需的。在釀酒酵母中，端粒由〜350 bp的雙鏈TG1-3重複DNA和〜12-14個核苷酸的單鏈TG1-3 DNA尾巴組成，以5`至3`方向延伸至染色體末端。我們和其他研究小組表明，Cdc13-Stn1-Ten1（CST）複合物與單鏈TG1-3 DNA結合以保護端粒。但是，CST複合物如何保護端粒的機制尚不清楚。本子計畫的重點是研究端粒如何保護染色體。擬藉由了解端粒的結構，來解釋該結構如何保護端粒免受降解，以及了解細胞如何區分端粒與雙鏈斷裂。在這裡，我們建議結合生物化學，分子遺傳學，單分子生物物理和結構生物學等方法來解決這些問題。具體的目標是： 目標1：通過單分子分析確定CST複合物的單鏈端粒DNA結合活性。 我們擬分析CST複合物的端粒結合特性。將使用FRET（螢光共振能量轉移）測定法來確定詳細的結合機理。特定的螢光團將被標記在DNA和/或CST蛋白上，然後使用FRET分析其構型變化。我們還將使用該測定法分析CST複合物在端粒上的結合。 目標2：使用低溫電子顯微鏡確定CST複合物的結構。 這項擬議研究的最終目標是了解CST如何保護端粒。解決CST端粒結構的結構將是最直接的方法。在這裡，我們建議應用低溫電子顯微鏡技術來解決這一生物學重要復合物的宏觀結構。 目標3：建立CST複合體的結構和功能關係。 我們將應用分子遺傳學方法在CST複合體上產生突變，然後分析其對端粒功能的影響。還將進行生化分析以評估這些突變體如何影響端粒結合特性。我們將評估Cdc13和CST複合物的生化特性。我們還將分析這些突變體如何調節端粒上的端粒轉錄RNA TERRA和R環。我們認為這種分析應該可以揭示端粒上CST複合物的結構和功能關係。" "Telomeres are the physical ends of eukaryotic chromosomes. They are required for the maintenance of chromosome integrity. In Saccharomyces cerevisiae, telomeres are composed of ~350 bp duplex TG1-3 repeated DNA and ~12-14 nucleotides single-stranded TG1-3 DNA tail running 5’ to 3’ to the end of chromosome. We and other groups have showed that the Cdc13-Stn1-Ten1 (CST) complex binds to the single-stranded TG1-3 DNA to protect telomeres. However, the mechanism of how CST complex protects telomeres is unclear. The focus of the proposed subproject is to study how telomeres protect and cap chromosomes. Specific questions addressed are to determine the structure of telomeres, how this specific structure protects telomeres from degradation, and to understand how cells differentiate telomeres from double strand breaks. Here we propose to combine biochemical, molecular genetics, single-molecule biophysical, and structural approaches to address these questions. Specific approaches are: Aim 1: To determine the single-stranded telomeric DNA binding activity of CST complex by single-molecule analyses. We propose to analyze the telomere binding properties of the CST complex. The detailed binding mechanism will also be determined using FRET (fluorescence resonance energy transfer) assays. Specific fluorophores will be tagged on DNA substrate and/or CST proteins and then analyzed for conformation alterations using FRET. We will also use this assay to analyze the binding of CST complex on telomeres. Aim 2: To determine the structure of CST complex using cryo-electron microscope. The ultimate goal of this proposed study is to understand how CST caps telomeres. Resolving the structure of CST-telomere structure will be the most direct approach. Here we propose to apply cryo-EM technique to solve the macrostructure of this biologically important complex. Aim 3: To establish structure and function relationship for CST complex. We will apply molecular genetics to generate mutations on CST complex and then assayed for their effects on telomere function. Biochemical analysis will also be conducted to evaluate how these mutants affect telomere binding properties. We will evaluate the biochemical properties of Cdc13 and the CST complex. We will also analyse how these mutant regulate telomere transcripts TERRA and R-loop on telomeres. We believe that this type of analysis should reveal the structure and function relationship of CST complex on telomeres."端粒染色體螢光共振能量轉移低溫電子顯微鏡TelomerechromosomeFRETcryoEM