臺灣博碩士論文加值系統

端粒(telomere)位於真核生物染色體末端，對於染色體的維持十分重要，在先前的研究顯示，小泛素修飾蛋白連接酶(SUMO E3 ligase)會影響端粒的機制，顯示細胞可能會藉由SUMO修飾端粒結合蛋白，影響蛋白功能來達到對端粒的調控，因此我們想要尋找可能受到SUMO修飾的端粒蛋白。SUMO修飾的蛋白質具有特定的共同序列，而Cdc13p具有兩個此特定序列，Cdc13p為必要的端粒結合蛋白，與許多端粒相關蛋白交互作用，並調控與維持端粒機制。利用純化的Cdc13p、Smt3p (SUMO)、E1及E2蛋白進行SUMO修飾反應，得知Cdc13p在in vitro 情況下會受到SUMO修飾。此外，利用親和性方式抓取在細胞大量表現的Smt3p-6His以偵測Cdc13p訊號，發現在in vivo時，Cdc13p亦會受到SUMO修飾。為了更加釐清此修飾對Cdc13p造成的影響，我將Cdc13p可能受到SUMO修飾的胺基酸進行點突變，利用in vivo及in vitro方式偵測，發現突變的cdc13p K909R、cdc13p K702R K909R 無法受到SUMO修飾，進而確定在Cdc13p中，K909為其SUMO修飾位。進一步觀察發現，分別表現此兩種突變蛋白的細胞，其端粒長度有些許延長的現象，而Cdc13p維持細胞存活的主要的功能、細胞內Cdc13p的含量及端粒位置效應並未受到改變。由以上結果得知，SUMO修飾會藉由調控端粒結合蛋白Cdc13p來影響端粒功能，顯示出SUMO修飾影響端粒功能的重要性。

Telomeres are specialized protein-DNA complexes at the end of eukaryotic chromosomes that play essential role in maintaining chromosome integrity. Protein factors that participate in telomere regulation and maintenance have been identified. They affect telomere function through their direct binding to telomeres, associate with telomere-binding proteins, or protein modifications. In Saccharomyces cerevisiae, both NFI1 and SIZ1 are involved in the last step of SUMO modification on proteins. We found that deletion of these two genes, nfi1Δ siz1Δ, caused longer telomeres, suggesting that SUMO modification might have an effect on telomeres. Interestingly, sequence analysis of telomere-binding protein, Cdc13p, revealed two putative sumoylation sites. Using purified E1, E2, and SUMO (Smt3p) in a SUMO modification assay, we found Cdc13p could be SUMO modified in vitro. Moreover, by pulling down SUMO with 6His Tag through Nickel-affinity chromatography, we also found that Cdc13p can be sumoylated in vivo. Furthermore, using site-specific mutagenesis to change the putative SUMO modification sites on Cdc13p, we found that the telomere lengths were affected in cells expressing these mutants. Thus, our results provide the first indication of SUMO modification on telomere binding protein Cdc13p and that SUMO modification might be a novel mechanism to regulate telomere functions.

論文電子檔著作權授權書…………………………………………… i
論文審定同意書…………………………………………………… ii
誌謝………………………………………………………………… iii
中文摘要…………………………………………………………… iv
英文摘要……………………………………………………………. v
目錄………………………………………………………………… vi
圖目錄……………………………………………………………… vii
緒論……………………………………………………………………… 1
材料方法………………………………………………………………… 7
實驗結果……………………………………………………………… 19
討論…………………………………………………………………… 25
參考文獻……………………………………………………………… 29
附圖表……………………………………………………………… 36

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