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研究生:莊孟哲
研究生(外文):Meng-Je Juang
論文名稱:以絲胺酸替換突變法分析阿拉伯芥Ku70蛋白之胱胺酸
論文名稱(外文):Analyzing Cysteine Residues of Arabidopsis thaliana Ku70 by Serine-Substituted Mutagenesis
指導教授:潘榮隆潘榮隆引用關係
指導教授(外文):Rong-Long Pan
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:45
中文關鍵詞:絲胺酸替換突變法
外文關鍵詞:cysteinesite-directed mutagenesis
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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.
Abbreviations…………………………………………………………….2


Abstract in Chinese……………………………………………………….4


Abstract …………………………………………………………………..6


Introduction ……………………………………………………………...8


Materials and Methods …………………………………………………13


Results ………………………………………………………………….20


Discussion ………………………………………………………………23


References………………………………………………………………25


Figures and Tables………………………………………………………32
Barik, S. (1997) PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering (White, B. A. eds) pp.173-182, Humana Press Inc., New Jersey, USA.
Bertuch, A. and Lundblad, V. (1998). Telomeres and double strand breaks: trying to make ends meet. Trends Cell Biol., 8, 339-342.
Blier, P. R., Griffith, A. J., Craft, J. and Hardin, J. A. (1993). Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. J. Biol. Chem., 268, 7594-7601.
Boubnov, N. V., Wills, Z. P. and Weaver, D. T. (1995). Coding sequence composition flanking either signal element alters V(D)J recombination efficiency. Nucleic Acids Res., 23, 1060-1067.
Bundock, P., Attikum, H. V. and Hooykaas, P. (2002). Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant. Nucleic Acids Res., 30, 3395-3400.
Critchlow, S. E. and Jackson, S. P. (1998). DNA end-joining: from yeast to man. Trends. Biochem. Sci., 23, 394-398.
de Vries, E., Driel, W. V., Bergsma, W. G., Arnberg, A. C. and van der Vliet, P. C. (1989). HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA multimeric protein complex. J. Mol. Biol., 208, 65-78.
Falzon, M., Fewell, J. W. and Kuff, E. L. (1993). EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J. Biol. Chem., 268, 10546-10552.
Francouer, A-M., Peebles, C. L., Compper, P. T. and Tan, E. M. (1986). Identification of Ki (Ku, p70/p80) autoantigenes and analysis of anti-Ki autoantibody reactivity. J. Immunol., 136, 1648-1653.
Gallego, M. E., Jalut, N. and White, C. I. (2003). Telomerase dependence of telomere lengthening in ku80 mutant Arabidopsis. Plant Cell, 15, 782-789.
Getts, R. C. and Stamato, T. D. (1994). Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J. Biol. Chem., 269, 15981-15984.
Giffin, W., Torrance, H., Rodda, D. J., Prefontaine, G. G., Pope, L. and Hache, R. J. G. (1996). Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature, 380, 265-268.
Griffith, A. J., Blier, P. R., Mimori, T. and Hardin, J. A. (1992). Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA. J. Biol. Chem., 267, 331-338.
Gu, Y., Jin, S., Gao, Y., Weaver, D. T. and Alt, F. W. (1997). Ku70-deficient embryonic stem cells have increased ionizing radition sensitivity, defective DNA end-binding activity and inability to support V(D)J recombination. Proc. Natl Acad. Sci. USA, 94, 8076-8081.
Mimori, T., Akizuki, M., Yamagata, H., Irada, S., Yoshida, S. and Homma, M. (1981). Characterization of a high molecular weight acdic nuclear protein recognised by autoantibodies from patients with polymyositis-schleroderma overlap. J. Clin. Invest., 68, 611-620.
Mimori, T. and Hardin, J. A. (1986). Mechanism of interaction between Ku protein and DNA. J. Biol. Chem., 261, 10375-10379.
Nussenzwieg, A., Chen, C., da Costa Soares, V. Sanchez, M., Sokol, K., Nussenzwieg, M. C. and Li, G. C. (1996). Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature, 382, 551-554.
Nussenzwieg, A., Sokol, K., Burgman, P., Li, L. and Li, G. C. (1997). Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival and development. Proc. Natl Acad. Sci. USA, 94, 13588-13593.
Okumura, K., Sakaguchi, G., Takagi, S., Naito, K., Mimori, T. and Igarashi, H. (1996). Sp1 family proteins recognize the U5 repressive element of the long terminal repeat of human T cell leukemia virus type Ⅰ through binding to the CACCC core motif. J. Biol. Chem., 271, 12944-12950.
Paillard, S., Strauss, F. (1991). Analysis of the mechanism of interaction of simian Ku protein with DNA. Nucleic Acids Res. 19, 5619-5624.
Rathmell, W. K. and Chu, G. (1994). A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol. Cell. Biol., 14, 4741-4748.
Reeves WH, Sthoeger ZM, Lahita RG. (1989). Role of antigen selectivity in autoimmune responses to the Ku (p70/p80) antigen. J. Clin. Invest., 84, 562-567.
Riha, K., Watson, J. M., Parkey, J. and Shippen, D. E. (2002). Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70. EMBO J., 21, 2819-2826.
Smider, V., Rathmell, W. K., Lieber, M. R. and Chu, G. (1994). Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science., 266, 288-291.
Taccioli, G. E., Cheng, H. L., Varghese, A. J., Whitmore, G. and Alt, F. W. (1994). A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation. J. Biol. Chem., 269, 7439-7442.
Tamura, K., Adachi, Y., Chiba, K., Oguchi, K. and Takahashi, H. (2002). Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks. Plant J., 29, 771-781.
Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N. W. Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zhang, J., Wang, S., Pongor, S. and Falaschi, A. (1994). Human DNA helicase Ⅱ -- a novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J., 13, 4991-5001.
Walker, J. R., Corpina, R. A. and Goldberg, J. (2001). Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature, 412, 607-614.
West, C. E., Waterworth, W. M., Story, G. W., Sunderland, P. A., Jiang, Q. and Bray, C. M. (2002). Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double-strand breaks in vivo. Plant J., 31, 517-528.
Yaneva, M., Wen, J., Ayala, A., Cook, R. (1989). cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J. Biol. Chem. 264, 13407-13411.
Yaneva, M. and Jhang, S. (1991). Expression of the Ku protein during cell proliferation. Biochim. Biophys. Acta., 1090, 181-187.
Yaneva, M., Kowalewski, T. and Lieber, M. R. (1997). Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies. EMBO J., 16, 5098-5112.
Yoo, S. and Dynan, W. S. (1998). Characterization of the RNA binding properties of Ku protein. Biochemistry, 37, 1336-1343.
Zhang, W. W., Yaneva, M. (1993). Reduced sulphydryl groups are required for DNA binding of Ku protein. Biochem. J., 293, 769-774.
Zhang, W. W., Yaneva, M. (1992). On the mechanisms of Ku protein binding to DNA. Biochem. Biophys. Res. Commun., 186, 574-579.
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