Ku蛋白於1981年由自體免疫缺失的病人血清中被發現，並證實為一種自體抗原，它是由兩個次體單元經非共價鍵結合而成的異分子雙體蛋白質，其分子量各為70道耳吞(Ku70)和80道耳吞(Ku80)。Ku蛋白是一種廣泛存在於細胞核內與去氧核糖核酸(DNA)結合之分子。當DNA在紫外光或自由基的作用下會產生末端斷裂，Ku蛋白主要會參與進行非同源DNA之末端結合，來修復雙股DNA的斷裂。在最近的研究中，已發現在高等植物阿拉伯芥中也發現Ku70和Ku80的同源基因(AtKu70和AtKu80)，並且它們與哺乳動物中的Ku具有類似的功能。不過比較不同的地方在於，Ku基因在哺乳動物中的表現量很高，而且是持續性的在細胞中表現；然而，在阿拉伯芥中發現的Ku基因雖在很多組織中都有表現，但其表現量卻非常低。
最近的研究指出，哺乳動物中的Ku蛋白與DNA結合需要其胺基酸序列中的還原態胱胺酸。另有研究指出，Ku70可以在沒有Ku80的存在下，單獨與DNA結合，但其結合能力比較微弱。為了探討Ku70上的那幾個胱胺酸與DNA之結合有關，我們使用絲胺酸替換突變法，將阿拉伯芥Ku70上的八個胱胺酸逐一替換成絲胺酸。所產生的突變株將被送到大腸桿菌內進行異體表現，並使用金屬親合性層析管柱(Ni2+-NTA)及高效率液相層析儀(FPLC)將蛋白質純化出來。利用十二脂鈉-聚丙醯凝膠電泳法(SDS-PAGE)和西方轉漬(Western blot)的分析，知道可以得到高純度的蛋白質。在此次研究中，我們以電泳層析分析實驗方法(Electrophoretic Mobility-shift Assay, EMSA)來分析突變的AtKu70蛋白質與DNA之結合能力。我們發現突變後的蛋白質只有C64S及C84S這兩株突變體還有活性，證實其餘六株突變體可能與DNA結合有關。未來我們將利用雙圓旋光譜儀(Circular Dichroism)等技術，進一步研究這些胺基酸殘基在Ku與DNA結合的角色。

Ku is a heterodimeric protein composed of two subunits of approximately 70 kDa (Ku70) and 80 kDa (Ku80). It is an abundant nuclear DNA-binding factor binding to double-stranded DNA ends without sequence specificity and is involved in DNA double strand breaks (DSBs) repair during ionizing radiation or free radical reaction. Recently, plant Ku protein has been isolated in 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, although several studies suggest that expression of Ku is dependent upon an entry into the cell cycle. In plants, AtKu gene is expressed widely and at low level in plant tissues, and probably induced during plant development.
In animals, it has been shown that the DNA-binding activity of Ku heterodimer depends on reduced cysteine residues in vitro, and the Ku70 subunit can weakly binds to the DNA end in the absence of Ku80. In this study, the 8 cysteine residues in AtKu70 were mutated by means of serine-substituted mutagenesis. Mutants were over-expressed in E. coli. The His-tagged fusion protein makes it possible to purify the soluble protein. With the application of immobilized-metal affinity column ,Ni2+-NTA (Ni2+-Nitriloacetic acid), and FPLC (Fast performance liquid chromatography) systems to purify the AtKu70, analysis by SDS-PAGE (Sodium dodecylsulphate - polyacrylamide gel electrophoresis) and Western blot reveal that the molecular weight of AtKu70 is about 70 kDa. According to the results of electrophoresis mobility shift assay (EMSA), we found that two mutants, C64S and C84S, have the DNA-binding activity. It indicated that the other 6 mutants could be involved in binding to DNA. In further studies, we will use the biophysical techniques, such as Circular Dichroism (CD), to investigate the structural and functional roles of these 6 mutants.

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