臺灣博碩士論文加值系統

端粒是真核染色體末端特化的由DNA以及蛋白組成的複合體，其功能參與在維持染色體的穩定性。在S. cerevisiae 中，端粒主要是由大約250-300 bp的 TG1-3/C1-3A雙股DNA 以及一段單股的TG1-3 DNA所組成，序列上具有重複性。端粒酶是真核細胞染色體末端複製時所需要的酵素，是一種ribonucleoprotein，在S. cerevisiae 中是由Tlc1 RNA 以及Est2p所組成，Est2p是具有反轉錄活性的蛋白質，Tlc1 RNA則是做為端粒酶複製時的模版，主要參與在端粒的複製以及長度維持。一些genetic證據顯示，端粒結合蛋白也會共同調控端粒酶的活性，例如Cdc13p、yKu複合體以及Est1p。Cdc13p是單股端粒DNA結合蛋白，並參與在端粒的保護以及端粒酶活性的調控上。雙股DNA末端結合蛋白yKu複合體是由yKu70和yKu80所組成，並且也會和Tlc1 RNA結合，同樣也參與在端粒長度的調控上。此外，Est1p是個會藉由和Cdc13p之間的互相作用而活化端粒酶的端粒相關蛋白。由於yKu複合體和Est1p的突變株都可以發現有端粒縮短的表現型，因此yKu複合體與Est1p可能在端粒長度的維持上扮演著正向的調控角色。為了進一步去分析這些端粒相關蛋白在分子機制上是如何去調控端粒酶的活性，因此，我們利用純化的Est2p/Tlc1 RNP複合體以及一個以端粒序列所設計模擬端粒結構的tailed-duplex DNA受質，已經在in vitro狀況下建立了偵測端粒酶活性的方法，以純化的端粒結合蛋白來分析其對端粒酶活性的調控。利用這樣in vitro的重建系統下，我們發現當Cdc13p或是yKu複合體結合至端粒上時，都會抑制端粒酶的活性，而Cdc13p所造成的端粒酶活性抑制是需要其端粒DNA結合能力。此外，Est1p可以回復Cdc13p對端粒酶活性所造成的抑制現象，顯示Est1p可能參與在活化端粒酶的活性。結合在in vivo genetic以及染色體免疫沈澱的實驗結果，可以提出一個在細胞週期的過程中，這些端粒結合蛋白如何去調控端粒酶活性的模型。

Telomeres are specialized protein-DNA structures at the physical ends of eukaryotic chromosomes. They are important for maintaining the stability of chromosomes. In yeast Saccharomyces cerevisiae, the telomere is composed of ~250-350 bp TG1-3/C1-3A double-stranded DNA and a single-stranded TG1-3 tail. Telomerase is a ribonucleoprotein involved in telomere replication and maintenance. Genetic evidence indicates that telomerase activity is regulated by telomere-associated protein factors, including Cdc13p, Ku proteins, and Est1p. Cdc13p is a single-stranded telomeric DNA binding protein that protects telomeres and regulates telomerase activity. Yeast Ku proteins, Yku70p and Yku80p, are the DNA end-binding proteins that bind telomere ends and telomerase Tlc1 RNA to regulate telomere length. Est1p is a telomerase-associated protein that activates telomerase through the interaction with Cdc13p. It appears that both Ku proteins and Est1p are positive regulators for telomere length maintenance as their mutations result in short telomeres. To elucidate the molecular mechanism of how these telomere-associated proteins affect telomerase activity, we established an in vitro telomerase analysis system using reconstituted telomerase core components, the template Tlc1 RNA and the catalytic subunit of Est2p. We have also synthesized a tailed-duplex DNA substrate made by telomeric DNA sequences to mimic telomere structure. Regulation of telomerase activity was then analyzed by adding the purified telomere-associated proteins. Using the in vitro reconstituted system, we found that binding of Cdc13p and/or Ku proteins to telomeres inhibited telomerase activity. The telomeric DNA binding activity of Cdc13p is required for its inhibition activity. Significantly, we found that Est1p was capable of re-activating the Cdc13p-inhibited telomerase activity, indicating a direct role of Est1p in activating telomerase activity. By combining the in vivo genetic and chromatin-immunoprecipitation results, a model for how telomere-associated proteins affect telomerase activity during cell cycle progression is proposed.

目 錄 1
圖表目錄 2
縮 寫 表 5
中文摘要 7
英文摘要 8
導 論 10
材料方法 17
實驗結果 28
討 論 38
參考資料 43
附 圖 49

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