運用單分子實驗探討端粒結合蛋白Cdc13及Est1在端粒上的交互作用

Abstract

Saccharomyces cerevisiae的端粒由TG1-3的重複DNA序列所組成，並帶有3’端的單股突出，而Cdc13可以結合至這樣單股TG1-3序列的DNA，避免染色體分解或被誤認為DNA斷裂。Cdc13也能透過和Est1交互作用來調控端粒酶的活性。為了瞭解詳細的調控機制，我們應用了單分子栓球試驗來進行分析，藉由單分子能夠觀測單一蛋白與DNA之間交互作用動態變化的特性，我們可以觀測Cdc13作用到端粒DNA受質上的動態變化。而我們發現當Cdc13結合到受質DNA後，會使受質DNA長度變短，同時觀察小球布朗運動值隨時間變化的關係圖，可以發現這些變化會停留在特定值且不同數值間能觀察到動態變化，而將這些這些特定布朗運動值換算成DNA長度，能得到Cdc13和DNA受質的不同構型間的DNA長度差值約等於70個鹼基對長，這些結果顯示了Cdc13能夠造成很大的構型變化，然而這些不同構型之間變化的調控機制尚未明瞭，因此我的論文第一部分是要分析Cdc13縮短DNA受質的詳細過程，像是反應過程是否需要多個Cdc13參與，而結果看來這樣的過程不需要多個Cdc13參與，而論文的第二部分則是要探討Est1是否會影響Cdc13結合DNA的能力或者Est1是否會影響Cdc13造成DNA受質縮短的能力，而結果顯示Est1會使Cdc13結合到DNA受質上的能力降低，但不會影響與DNA形成二級結構的Cdc13結合在DNA受質上的能力。

The telomeres of Saccharomyces cerevisiae consist of TG1-3 repetitive DNA sequences with a 3’ single strand overhang. Cdc13 binds to the single-strand TG1-3 DNA to prevent telomeres from degradation and from being recognized as DNA damages. Cdc13 also interacts with Est1 to regulate telomerase activity. To understand the detail regulatory mechanism, we applied single molecular analysis, the tethered particles motion (TPM) assays, to monitor the dynamic interaction between Cdc13 and DNA substrate. We found that the DNA length was shortened upon binding by Cdc13. Significantly, time tracing experiments found that DNA length was dynamically changed in a stepwise manner with each step equal to ~70 bps. The results suggested that Cdc13 caused a large and distinct conformational change on DNA. However, the mechanism of how Cdc13 shortens DNA is unclear. Here I analyzed whether telomere shortening requires multiple molecules of Cdc13. The results implicated that multiple molecules of Cdc13 was not required to induce DNA shortening by Cdc13. I also tested whether Est1 affected the binding and shortening of DNA by Cdc13. The results showed that Est1 decreased the binding of Cdc13 to telomeric DNA, however, Est1 did not affect the binding of Cdc13 in the form of shortened DNA.