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研究生:瞿立威
研究生(外文):Li-Wei Chu
論文名稱:DNA末端特性影響Ku蛋白直接移轉能力之研究
論文名稱(外文):The studies of Ku direct transfer influenced by the characteristics of DNA ends
指導教授:周文剛
指導教授(外文):Wen-Gang Chou
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:69
中文關鍵詞:Ku蛋白非同源性末端連結DNA雙股斷裂
外文關鍵詞:Ku proteinnon-homologous end joiningDNA double strand break
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在哺乳動物細胞中,存在著兩種主要的DNA雙股斷裂修補機制,一種是同源性重組作用(homologous recombination),另一種則是非同源性末端連結作用(non-homologous end joining)。而在整個細胞週期的運行中又以非同源性末端連結作用最常被使用來進行DNA雙股斷裂的修補。在非同源性末端連結的修補機制中,DNA依存性蛋白質激脢(DNA-PK)擔負著非常重要的責任,而Ku蛋白則是這個蛋白質激脢聚合體中必要的單體。Ku蛋白是由Ku70及Ku80兩個次單元所構成的雜二次體,能夠與DNA末端結合促使DNA依存性蛋白質激脢催化單元(DNA-PKcs)的活化,進而徵集ligase Ⅳ與XRCC4 (X-ray repair cross complementing protein 4)對DNA雙股斷裂進行修補。故對於哺乳動物細胞而言,Ku蛋白及DNA-PKcs的變異或缺失往往是導致DNA雙股斷裂修補失敗以及輻射敏感作用的主因。
在先前的研究當中發現,Ku蛋白除了DNA末端的結合能力以外,亦能夠由一個DNA分子直接移轉至另一個DNA分子。有趣的是,此種直接移轉的發生並非Ku蛋白脫離原先的DNA再結合另一個DNA;而是藉由DNA-Ku-DNA聚合體的形成,Ku蛋白於二DNA末端間直接移轉過去。在本論文的研究當中,特別設計不同末端構型的DNA來研究Ku蛋白在二DNA間直接移轉的能力。經由競爭性電泳膠泳動實驗分析,發現到在低鹽環境(50 mM NaCl, 10mM MgCl2)下二DNA末端突出序列互補是Ku蛋白發生直接移轉的重要條件,末端平鈍構型的DNA不具備突出單股序列,故無法滿足末端突出序列互補的條件使Ku蛋白直接移轉失敗;而在高鹽環境(200 mM NaCl, 5mM MgCl2)的實驗中,Ku蛋白則能夠於不同的DNA末端之間直接移轉,例如由5’突出末端直接移轉至平鈍或3’突出末端。此外在二DNA末端突出序列互補時,Ku蛋白只能由特殊dA•dT聚合片段移轉至一般的DNA片段,而無法由一般的DNA片段上直接移轉至特殊dA•dT聚合片段。但是增加一般的DNA序列於特殊dA•dT聚合片段中時,Ku蛋白便能由一般DNA片段直接移轉到這個dA•dT聚合片段上。故推測低鹽環境下DNA末端突出序列的互補將幫助DNA-Ku-DNA構型的形成,進而幫助Ku蛋白在兩段DNA間的直接移轉;而高鹽環境下DNA-Ku-DNA構型的形成則不需要DNA末端突出序列的互補。當DNA-Ku-DNA構型形成後,Ku蛋白由DNA-Ku-DNA構型移轉至其一DNA的能力將受到DNA雙股序列穩定性的影響,而Ku蛋白將停留於較為穩定的DNA雙股片段上。
In mammalian cells, there are two different pathways for repairing DNA double strand breaks (DSBs). One is homologous recombination, and the other is non-homologous end joining. In mammalian cells, DSBs repair proceeds predominantly by non-homologous end joining. In non-homologous end joining pathway, DNA-dependent protein kinase (DNA-PK) takes on an important responsibility. Ku protein is a subunit in DNA-PK. Ku protein is a heterodimer composed of Ku70 and Ku80. Ku protein can interact with double-stranded DNA ends. Ku protein binds DNA ends and recruits the catalytic subunit of DNA-PK (DNA-PKcs) to DNA in order to assemble an active complex. DNA-PK active complex recruits DNA ligase Ⅳ and XRCC4 to repair DSBs. In mammalian cells, deficiency of either Ku protein or DNA-PKcs is the major reason to cause DNA double strand break repair failure.
In this study, we demonstrated that Ku protein could not only bind DNA ends but also can transfer between two DNA molecules. The transfer of Ku is not to release from one DNA and bind to another DNA. Instead, Ku protein can transfer directly between two DNA molecules with homologous cohesive ends. Besides, increasing concentration of sodium chloride in the reaction, Ku protein can transfer between DNA with non-homologous ends. However, Ku protein can not transfer from DNA with random sequence (general DNA molecule) to a poly-dAdT fragment even with homologous cohesive ends. But, Ku protein can transfer from a poly-dAdT fragment to a general DNA molecule with homologous cohesive ends. Interestingly, increasing random DNA sequence in the poly-dAdT fragment, we observed Ku protein can transfer to the poly-dAdT fragment. In conclusions, we suggest from these results that there are two steps in the direct transfer of Ku protein. The first step is the formation of a DNA-Ku-DNA complex. In this step, it is required that the two DNA molecules have homologous cohesive ends under low salt condition but not under high salt condition. In the second step, Ku protein moves from one DNA molecule to another one. In this step, the dinection of movement of Ku under certain situation is influenced by DNA sequences.
目錄 …………………………………………………… Ⅰ
中文摘要 …………………………………………………… Ⅱ
英文摘要 …………………………………………………… Ⅳ
引言 …………………………………………………… 1
材料與方法 …………………………………………………… 13
結果 …………………………………………………… 21
討論 …………………………………………………… 37
參考文獻 …………………………………………………… 47
圖表 …………………………………………………… 52
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