中文摘要
Ku蛋白首先發現於1981年自體免疫缺失的病人血清中，並且證實其為一種自體抗原，它是由兩個次體單元經由非共價鍵結合而成的異分子雙體蛋白質，其分子量分別為70道耳吞(Ku70)和80(Ku80)道耳吞。Ku蛋白是一種廣泛存在於細胞核內與去氧核醣核酸(DNA)結合之分子。當DNA暴露在紫外光或者經由自由基的作用下會產生末端斷裂，而Ku蛋白主要功能即為參與非同源DNA之末端的結合，以修復雙股DNA的斷裂。在最近的研究中，發現在高等植物阿拉伯芥中也有Ku70和Ku80的同源基因(AtKu70和AtKu80)，其功能與哺乳動物中的Ku具有類似的功能。但是比較不同的地方在於，Ku基因在哺乳動物中的表現量很高，且是持續性在細胞中表現，然而在阿拉伯芥中,AtKu基因雖然在許多組織都有表現，但是其表現量卻非常低。
最近的研究也指出，哺乳動物中的Ku蛋白是藉由其胺基酸序列中的還原態胱胺酸來與DNA結合。另外也有研究指出，Ku70可以在沒有Ku80的存在下，單獨地與DNA結合，只是其結合力較微弱。所以為了探討AtKu70是藉由哪幾個胱氨酸與DNA結合，我們使用了絲胺酸替換突變法，將阿拉伯芥Ku70上的八個胱胺酸逐一由絲胺酸取代。接著將突變株送至大腸桿菌進行異體表現，並使用金屬親合性層
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析管柱(Ni2+-NTA)及高效率液相層析儀(FPLC)將蛋白質純化出來。在這次的研究中，我們用電泳遷移率檢測 (Electrophoresis mobility shift assay) 以及圓二色性光譜儀Circular dichroism (CD) spectrometer，去分析突變的AtKu70與DNA的結合力以及去觀察其二級結構。在這次研究中，我們發現突變後的蛋白除了C43S外，其餘的DNA結合利皆下降，尤其以C46S以及C64S下降最多，證實了這兩個位置的胱氨酸與DNA結合有關。

Abstract
Ku is a heterodimeric protein composed of two subunits known as Ku70 (70 kDa) and Ku80 (80 kDa), respectively. It is an abundant nuclear DNA-binding factor binding to double-stranded DNA ends without sequence specificity. After ionizing radiation or free radical reaction Ku is involved in DNA double strand breaks (DSBs) repair through NHEJ pathway. Recently, plant Ku protein has been isolated from Arabidopsis thaliana, and its functions are similar to those of mammalian cells. However, the expression of Ku gene is constitutively at high levels in mammalian cells while the concentration of AtKu protein is relatively lower in humans.
In recent study, Ku protein binding to DNA end in mammalian cells depends on its reduced cysteine residues, and Ku70 can weakly bind to the DNA end alone in the absence of Ku80. In order to determine which cysteine residue of AtKu70 binds to DNA, all 8 cysteine residues in AtKu70 were mutated by serine-substituted mutagenesis. Mutants were expressed in E. coli and purified by immobilized-metal affinity column, Ni2+-NTA and FPLC system. In this study, according to the results of electrophoresis mobility shift assay (EMSA) and circular dichroism (CD) spectrometry, we found that all mutants except C43S decreased their binding affinity, especially C46S and C64S decline significantly. The results indicate that these two cysteine residues are directly responsible for DNA binding capacity.

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