2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643316摘要：端粒(telomere)是位於真核生物線性染色體末端的DNA 和蛋白質複合體，能防止有害的染色體重組及維護染色體穩定性，進而避免發生與其相關的癌症。端粒是由G-rich 簡單的重複序列和用來延伸其長度的端粒酶(telomerase)所組成。當酵母菌和人類細胞缺乏端粒酶時，端粒長度會隨著複製的次數漸增而逐漸縮短，最後則進入老化的狀態。有一些蛋白附著於端粒上，其中一些蛋白能調控端粒酶的活性，另外一些則是與染色體末端的保護有關。Cdc13p 在這些端粒功能中具有重要的貢獻，它具有高度序列專一性並附著於單股端粒DNA 上。Cdc13 藉由吸引端粒酶到端粒進行端粒複製，並藉由附著在端粒的單股DNA 上來達到保護染色體末端的功能。因為哺乳動物細胞中的端粒也具有相似的結構及功能，所以相同的機制在人類細胞也可能是被需要的。本計畫的長期目標是藉由研究Cdc13 的磷酸化進而探討端粒複製和端粒維持的機制。在之前的研究中，我們發現端粒在in vivo 進行重組時需要Cdc13、和檢查點磷酸化酵素Mec1 及Tel1 三個蛋白質作用。我們又進一步發現Mec1 及Tel1 可以磷酸化Cdc13。這對於端粒酶在cell cycle S 時期的晚期能被吸引至端粒是很關鍵的步驟。為了想深入探討端粒酶活化時其訊息傳導的機制，我們將研究在cell cycle S 時期的晚期中，Cdc13 如何吸引Mec1 和Tel1 磷酸化酵素至端粒以及這樣的訊息如何在cell cycle M 時期被終止。這項計畫的主要目標如下:1. 決定Mre11 是否會吸引Tel1 到短端粒的Cdc13 上2. 決定Ddc2 是否會吸引Mec1 到短端粒的Cdc13 上3. 檢查Rsp5 所引起的Cdc13 ubiquitination 是否能移除端粒上磷酸化的Cdc13 進而停止端粒複製4. 檢查去磷酸酶(phosphatase)所引發的Cdc13 去磷酸化是否能消除磷酸化的Cdc13執行吸引端粒酶的功能由於人類細胞也具有和Cdc13功能相似的同源蛋白質POT1，完成此計畫能讓我們進一步了解酵母菌和人類的癌症細胞中端粒複製的機制。在癌細胞的增殖中端粒長度的維持是一個重要的步驟，因此本研究的結果將會和如何調控癌症細胞的端粒長度息息相關，對於癌症的治療更是一大貢獻。<br> Abstract: Telomeres are dynamic DNA-protein complexes that protect the ends of linearchromosomes, prevent detrimental chromosome rearrangements and defend againstgenomic instability and the associated risk of cancer in eukaryotic cells. Telomeres arecomposed of telomeric DNA, consisting of tandem repeats of short G-rich sequences,and are synthesized by the enzyme telomerase. In both yeast and human cells whentelomerase activity is absent, telomeres are gradually shortened and it eventually causesreplicative senescence. Telomeres are capped by many binding proteins. Some telomericproteins regulate the telomerase activity, while others participate in the protection ofchromosome ends. Cdc13p is a central player to several aspects of telomeric functions. Itexhibits sequence-specific binding for single-stranded telomeric DNA. Cdc13p mediatesboth telomere replication by recruiting telomerase and performs an essential function inprotection of telomeres. Since mammalian telomeres also contain similar structure andfunctions, similar mechanisms may be required in human cells.The long term objective of this study is to elucidate the mechanism of telomerereplication and maintenance through the study of Cdc13p phosphorylation. We previouslydemonstrated that Cdc13p and checkpoint kinases, Mec1p and Tel1p, are all required forproper telomere recombination in vivo (Tsai et al., 2002, MCB). We further found thatCdc13p can be phosphorylated by Mec1p and Tel1p (Tseng et al., 2006, NAR). This is acritical step to recruit telomerase to telomere at the late S phase. We are interested in a deepermechanistic understanding regarding the signal transduction pathway of telomerase activationby pursuing how Cdc13p recruit Mec1 and Tel1 kinases to telomeres at late S phase and howthis signal is turned off at M phase. The specific aims for this grant are:1. To determine whether Mre11 recruits Tel1 to Cdc13 on short telomeres2. To determine whether Ddc2 recruits Mec1 to Cdc13 on short telomeres3. To examine if Rsp5-mediated ubiquitination of Cdc13 provides a mechanism toeliminate phosphorylated Cdc13 from telomeres for posttelomeric processes4. To examine if phosphatase-mediated dephosphorylation of Cdc13 contributesto elimination of phosphorylated Cdc13 from recruiting telomeraseAccomplishment of this project will advance our knowledge on the mechanism oftelomere replication in both yeast and human cancers, given that human contains a Cdc13portholog named POT1. Telomere maintenance is a critical step in cancer formation. Therefore,the results from this study will be relevant to how to regulate telomere lengths in cancer cellsand these findings will have significant contribution to the treatment of cancer.端粒DNA 複製DNA 修復癌症酵母菌TelomereDNA replicationDNA repairCancerYeast