臺灣博碩士論文加值系統

摘要
本實驗室先前發現斑馬魚受精12小時後的胚胎蛋白粗萃取液(12 hpf CE, crude extracts of 12 hr after fertilization) 對於受到紫外線傷害的DNA有高度的辨識活性，並且將凝膠阻抗實驗膠片上的DNA-protein複合體之小點挖下，經質譜鑑定的分析，發現和150kDa的斑馬魚卵黃前質1蛋白質 (vitellogenin Ⅰ, zfVg 1)有高度的相似性 (Lai et al., 2006)。一般真核生物的NER模式是利用辨識蛋白結合至DNA受損的位置後，藉由ATP的水解才能進行後續的切割及修復等作用，故可以得知ATP在NER機制中所扮演的重要性。因此先前實驗亦探討12 hpf CE 有無NER過程中切割蛋白的活性，發現於2mM ATP的環境下，12 hpf CE對於受紫外線傷害的質體DNA有較高的切割專一性。
為了要再深入探討12 hpf CE是否因為ATP的水解而影響辨識蛋白在DNA受損位置上的結合，所以本論文中進一步將ATP和12 hpf CE 做反應後，利用帶有6-4PP之TC探針做為凝膠阻抗實驗 (EMSA) 的受質，結果發現隨著ATP濃度的增加，DNA-protein複合體之訊號強度會逐漸減弱，甚至在2mM ATP時，其訊號完全消失。而在二維蛋白電泳的結果中，發現ATP會造成12 hpf CE 的蛋白組成改變。另外再以ATP-γ-S及ADP取代ATP來進行EMSA實驗，發現濃度為2mM時可以直接觀察到其訊號有部分的減弱但不至於消失。因此選擇ATP-γ-S取代ATP和12 hpf CE 反應後，再進行二維蛋白電泳，從實驗結果中可以發現12 hpf CE 中有一群蛋白質會因著ATP的存在而發生組成的改變；另外的一群蛋白質會受到ATP的水解而造成蛋白質的降解。另外藉由蛋白質磷酸根染色的實驗中，可以發現藉由2mM ATP和12 hpf CE反應一分鐘後，即會有蛋白質受到磷酸根修飾的情形。為了得知是否因為ATP的酸鹼值過酸，導致辨識蛋白的降解以至於EMSA凝膠上的訊號消失，因此利用20mM Tris-HCl，將其調整不同酸鹼值 (pH2-8) 後，再與EMSA實驗的反應物混合後，發現在低酸鹼值時，雖然訊號有減弱的現象，但不至於會造成訊號的消失。為了找出是那些的蛋白質會受到2mM ATP的影響，利用蛋白質激酶抑制劑，gefitinib和EMSA反應物混合後進行實驗，發現gefitinib無法使受到抑制的DNA-protein複合體之訊號回復。另外以陰離子交換樹酯將12hrs CE做分離、純化後，將不同電性的蛋白質進行EMSA實驗，發現帶負電荷的蛋白質對於帶有6-4PPs之TC探針會形成DNA-protein的複合體；但正電荷的蛋白質卻無辨識結合受損DNA的專一性。進而再以2mM ATP分別與純化出的蛋白質做反應後，進行相同的實驗，發現無論是帶何種電性的蛋白質皆會受到2mM ATP的影響，造成DNA-protein複合體之訊號消失。由以上的實驗得知12 hpf CE 可能受到ATP水解的影響，造成12 hpf CE 當中的組成改變，但12 hpf CE 中究竟是那ㄧ種蛋白質會受到ATP水解的影響，仍需進一步的研究。

Abstract
In most organisms, cyclobutane pyrimidine dimmers (CPDs) and (6-4) photoproducts (6-4 PPs) produced after UV irradiation are removed from damaged DNA primarily by nucleotide excision repair (NER) that introduces a dual incision at both ends of the lesion after an ATP-dependent DNA zebrafish (Danio rerio) vitellogenin 1 (zfVg1) had been identified as the components of a UV-damaged-DNA binding activity highly expressed in zebrafish embryos at 12 hr post fertilization (hpf) (Lai et al., 2006). Because of the requirement of ATP for DNA unwinding during NER, the effects of ATP on the binding of zfVg1-like proteins to 6-4PPs were examined in this study. The binding of 12 hpf zebrafish extract proteins to a 6-4 PP probe was suppressed by 2mM ATP and partially inhibited by ATP-γ-S at the same concentration. Hence, 6-4PP-dependent binding suppressed by ATP was mediated through ATP hydrolysis. The pH change induced by ATP addition was unable to produce the suppressive effect. The inability of ATP-treated extracts to bind 6-4PPs correlated well with the disappearance of a few 70 to 100-kDa zfVg1-like polypeptides electrofocused at pH about 3.5 and the appearance of novel 33 to 90-kDa zfVg1-like factors having neutral to weak basic pIs, indicating the importance of high-molecular-weight zfVg1-like factors in maintaining the UV-binding activity except the two 30 to 35 kDa polypeptides. High and low-molecular-weight zfVg1-like proteins contained in zebrafish extracts might contact UV-damaged DNA as protein complexes after immunoblot analysis of UV-binding protein fractions. ATP was found to disturb UV-dependent binding via protein phosphorylation induced 1 min after ATP addition. The physiological role of this ATP-induced suppression of UV-dependent binding awaits further investigation.

目錄
中文摘要 1
英文摘要 3
前言 4
材料與方法
1、 收集斑馬魚胚胎 7
2、 斑馬魚胚胎萃取液之製備 7
3、 蛋白質定量 8
4、 離子交換層析法 8
5、 凝膠阻抗實驗 9
6、 SDS-PAGE蛋白質分析 10
7、 二維蛋白電泳分析 11
8、 SYPRO-Ruby stain 11
9、 Phosphoprotein stain 12
10、 西方氏點墨法 12





結果
1、 ATP對於斑馬魚胚胎蛋白與DNA傷害結合 14
2、 ATP誘發Vg1類似蛋白成份改變之二維電泳分析 15
3、 ATP、ATP-γ-S及ADP抑制12hpf CE之結合能力 15
4、 12hpf CE受到ATP-γ-S影響之二維電泳分析
16
5、 ATP水解造成12 hpf CE中的蛋白質受到磷酸化之情形 16
6、 環境酸鹼值的不同對於Vg1類似蛋白結合至6-4PP探針之影響 17
7、 gefitinib回復Vg1類似蛋白─6-4PP複合體形成之能力
17
8、 不同電性蛋白質辨識結合6-4PP之專一性差異
18
9、 不同電性蛋白質之免疫分析
19
10、 單株斑馬魚抗體抑制帶負電荷蛋白質結合6-4PP探針之影響
19
11、 ATP抑制不同電性蛋白質對於DNA的結合
19
討論 21
參考文獻 26
圖、表 30

參考文獻
Anderson, T. A., Levitt, D. G. and Banaszak, L. J. (1998). The Structural Basis of Lipid Interactions in Lipovitellin, a Soluble Lipoprotein. Structure 6, 895-909.
Birsoy, B., Kofron, M., Schaible, K., Wylie, C. and Heasman, J. (2006). Vg 1 is an Essential Signaling Molecule in Xenopus Development. Development 133, 15-20.
de Laat, W. L., Appeldoorn, E., Sugasawa, K., Weterings, E., Jaspers, N. G. J. and Hoeijmakers, J. H. J. (1998). DNA-Binding Polarity of Human Replication Protein A Positions Nucleases in Nucleotide Excision Repair. Genes Dev. 12, 2598-2609.
de Laat, W. L., Jaspers, N. G. J. and Hoeijmakers, J. H. J. (1999). Molecular Mechanism of Nucleotide Excision Repair. Genes Dev. 13, 768-785.
Fagotto, F. (1990). Yolk Degradation in Tick Eggs: I. Occurrence of a Cathepsin L-Like Acid Proteinase in Yolk Spheres. Arch. Insect Biochem. Physiol. 14, 217-235.
Hiramatsu, N., Matsubara, T., Hara, A., Donato, D., Hiramatsu, K., Denslow, N. and Sullivan, C. (2002a). Identification, Purification and Classification of Multiple Forms of Vitellogenin from White Perch (Morone Americana). Fish Physiol. Biochem. 26, 355-370.
Hiramatsu, N., Ichikawa, N., Fukada, H., Fujita, T., Sullivan, C. V. and Hara, A. (2002b). Identification and Characterization of Proteases Involved in Specific Proteolysis of Vitellogenin and Yolk Proteins in Salmonids. J. Exp. Zool. 292, 11-25.
Hsu, T., Cheng, C., Shih, C. and Yeh, F. (2001). Detection and Partial Characterization of a UV-Damaged-DNA Binding Activity Highly Expressed in Zebrafish (Danio Rerio) Embryos. Fish Physiol. Biochem. 25, 41-51.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of Embryonic Development of the Zebrafish. Dev. Dyn. 203, 253-310.
Lai, Y. S., Chiue, L. F. and Hsu, T. (2006). Low-Molecular-Weight Vitellogenin 1-Like Proteins are Components of a UV-Damaged-DNA Binding Activity Highly Expressed in Zebrafish (Danio Rerio) Embryos. J. Exp. Zoolog A. Comp. Exp. Biol. 305, 215-224.
Lehmann, A. R. (2003). DNA Repair-Deficient Diseases, Xeroderma Pigmentosum, Cockayne Syndrome and Trichothiodystrophy. Biochimie 85, 1101-1111.
Paria, B. C., Tsukamura, H. and Dey, S. K. (1991). Epidermal Growth Factor-Specific Protein Tyrosine Phosphorylation in Preimplantation Embryo Development. Biol. Reprod. 45, 711-718.
Patrick, S. M. and Turchi, J. J. (1999). Replication Protein A (RPA) Binding to Duplex Cisplatin-Damaged DNA is Mediated through the Generation of Single-Stranded DNA. J. Biol. Chem. 274, 14972-14978.
Sancar, A. (1995). Excision Repair in Mammalian Cells. J. Biol. Chem. 270, 15915-15918.

Sarker, A. H., Tsutakawa, S. E., Kostek, S., Ng, C., Shin, D. S., Peris, M., Campeau, E., Tainer, J. A., Nogales, E. and Cooper, P. K. (2005). Recognition of RNA Polymerase II and Transcription Bubbles by XPG, CSB, and TFIIH: Insights for Transcription-Coupled Repair and Cockayne Syndrome. Mol. Cell 20, 187-198.
Seidl, C. and Moritz, K. B. (1998). A Novel UV-Damaged DNA Binding Protein Emerges during the Chromatin-Eliminating Cleavage Period in Ascaris Suum. Nucleic Acids Res. 26, 768-777.
Seki, N., Hayashi, A., Hattori, A., Kozuma, S., Sasaki, M., Suzuki, Y., Sugano, S., Muramatsu, M. and Saito, T. (1999). CDNA Cloning, Tissue Expression, and Chromosomal Assignment of a Mouse Gene, Encoding a 127 kDa UV-Damaged DNA Binding Protein which is Defective in XPE Cells. DNA Res. 6, 319-322.
Thiagalingam, S. and Grossman, L. (1993). The Multiple Roles for ATP in the Escherichia Coli UvrABC Endonuclease-Catalyzed Incision Reaction. J. Biol. Chem. 268, 18382-18389.
Wang, H., Yan, T., Tan, J. T. and Gong, Z. (2000). A Zebrafish Vitellogenin Gene (vg3) Encodes a Novel Vitellogenin without a Phosvitin Domain and may Represent a Primitive Vertebrate Vitellogenin Gene. Gene 256, 303-310.
You, J. S., Wang, M. and Lee, S. H. (2003). Biochemical Analysis of the Damage Recognition Process in Nucleotide Excision Repair. J. Biol. Chem. 278, 7476-7485.

賴意繡(2005).小球藻與斑馬魚胚胎中DNA傷害辨識蛋白之分離與鑑定.國立台灣海洋大學生物科技所博士論文.81-98.
謝豐如(2006).斑馬魚早期胚胎中卵黃前質1類似蛋白對紫外光傷害DNA的辨識及切割作用.國立台灣海洋大學生物科技所碩士論文.