2013-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643237摘要：端粒（telomere）為真核生物染色體末端的特殊結構，由DNA及結合性蛋白所組成的複合物。其作用為保護染色體末端避免被分解及幫助染色體完整複製。端粒酵素（telomerase）是一個含有RNA與蛋白質的椱合體，它利用其RNA為模版來延長端粒序列，為端粒長度維持所必須。包括了Cdc13p，Yku，Est1p在內的端粒結合蛋白質一起參與在端粒酵素活性的調節。其中Cdc13p與Yku可直接與端粒序列結合，Est1p雖不能直接與端粒序列結合，但是它與端粒酵素結合藉以調節端粒酵素活性。雖然已有許多遺傳實驗證明這幾個蛋白質參與端粒長度維持，然而其分子機制並不明確。本實驗室初步利用純化的蛋白質，成功的利用純化的核心端粒酵素（Est2p/Tlc1 RNA）建立了端粒酵素活性分析系統。於此、本計畫將結合蛋白質生化分析與細胞生物學的方法，擬利用純化的核心端粒酵素作實驗，探討這些發現粒結合蛋白質如何調節端粒酵素活性；並以螢光蛋白質（YFP、CFP）接上端粒蛋白質，直接在顯微鏡下以螢光觀察這些蛋白質之間的交互作用。端粒結合蛋白質Cdc13p除了影響端粒酵素之外，亦會藉由直接與DNA複製酵素Pol1p結合，參與端粒長度的維持。由於實驗初步研究結果顯示Cdc13p可能藉由形成雙複合體來調節其與Pol1p的結合。本計畫亦擬進一步分析Cdc13p與Pol1p結合對端粒影響的分子機制，釐清其在端粒的角色。由於端粒長度的維持在不同生物物種之間保留性極高，因此本計畫研究成果將有助於瞭解端粒對於老化與癌症細胞維持的影響。<br> Abstract: Telomeres, the DNA-protein complexes located at the ends of most eukaryotes, protect chromosome ends form nucleolytic digestion and facilitate complete replication of the genome. Telomerase is a ribonucleoprotein (RNP) that utilizes its template RNA to extend telomeric G-strand DNA sequences. It is regulated by telomere proteins including Cdc13p, Est1p, and Yku protein. Although genetic evidence suggested that these proteins are required for proper function of telomerase activity, the mechanism of how these proteins affect telomerase activity is less clear. Here we propose to apply both biochemical and cell biological analyses to address how these proteins affect telomerase. The reconstituted system using core telomerase RNP (Est2p/Tlc1 RNA) will be applied in this study. Recombinant telomere-associated proteins will also be isolated and used to test its effects on telomerase. Fluorescent fusion proteins will be generated that fuse an YFP or CFP to telomere proteins. The interactions between telomere proteins and telomerase will then be observed directly under a fluorescent microscope. Cdc13p also mediates C-strand synthesis through interacting with DNA polymerase  (Pol1p). The role of Cdc13p-Pol1p interaction on telomere function will also be analyzed. Since the mechanisms underlying telomere length maintenance are highly conserved among eukaryotes, results from this study should help us understand the mechanisms of aging and cancer.端粒染色體telomerechromosome