臺灣博碩士論文加值系統

中文摘要
DNA 受紫外線C 照射後所產生之雙嘧啶產物，主要由核酸切割修補
作用 (nucleotide excision repair, NER) 加以修復；而本實驗室先前已証實斑馬魚早期胚胎中缺乏類似已知NER 分子如XPA 之蛋白(25)，而含有兩個分子量約為30 與35 kDa 且胺基酸序列類似150 kDa Vitellogenin1 (Vg1) 之DNA 傷害辨識蛋白。本論文目的在繼續探討斑馬魚胚胎中是否含有其他結構類似Vg1 之DNA 傷害辨識蛋白及類似Vg1 之辨識蛋白是否可能參與NER。實驗發現製備級蛋白質等電點分離系統能有效的將受精12 小時後斑馬魚胚胎中之DNA 傷害辨識蛋白進行分離。凝膠阻抗實驗顯示等電點約3-6 之蛋白無法區分正常與受UV 傷害之DNA，但等電點約7-9 之蛋白對於受UV 傷害DNA 具明顯辨識能力，而且依據各蛋白分離液對金屬螯合劑1, 10-phenanthroline (OP) 之不同反應發現斑馬魚胚胎中至少含有兩群
類似Vg1 之UV 傷害辨識蛋白。凝膠阻抗試驗發現等電點約 6.2-6.5 之分離液中含有之前發現的 35 kDa Vg1 類似蛋白，其辨識紫外線傷害DNA 之能力受OP 抑制，然而等電點 7-8 之分離液不含上述之35 kDa 蛋白，且DNA 傷害辨識能力受OP 之作用而明顯增加，而先前實驗已發現其中含有分子量25 kDa 之Vg1 類似蛋白(35)，因此斑馬魚胚胎中應含有兩群類似Vg1 之紫外線傷害辨識蛋白。
為了解Vg1 類似蛋白除了辨識外是否亦會切割或修補紫外線傷害
DNA(36)，本論文也進行核酸切割修補試驗，以pGEM-T 超螺旋質體作為DNA 切割實驗之受質，觀察開放型質體生成量以確認切割能力(37)。斑馬魚胚胎萃取液在10-20 µg 蛋白量並在有ATP 存在下，對紫外線傷害DNA具有專一性切割能力。加入抗150 kDa Vg1 抗體發現可以抑制斑馬魚胚胎萃取液對紫外線傷害DNA 之專一性切割能力，使胚胎萃取液對未照射DNA 之切割能力上升，顯示Vg1 類似蛋白對紫外線傷害DNA 具有辨識能力。斑馬魚胚胎萃取液經等電點分離液後，發現在中性範圍對紫外線傷害DNA 均有微弱切割活性，表示胚胎萃取液中辨識與切割蛋白可能為一種複合體。

ABSTRACT
Dipyrimidine photoproducts induced by UV irradiation on DNA are primarily removed from UV-damaged DNA by nucleotide excision repair (NER). NER proteins homologous to the human XPA were not detected in the extracts of zebrafish (Danio rerio) embryos, yet these extracts were previously found to contain two 30 to 35 kDa UV-damaged-DNA binding proteins
homologous to the 150 kDa vitellogenin1 (Vg1). The objectives of this research were to examine if multiple Vg1-like UV-binding factors existed in zebrafish embryos and whether these factors were involved in NER. Nonspecific and UV-specific DNA binding molecules present in 12-hr-old
zebrafish embryos were efficiently separated by a reparative
isoelectrofocusing. Two forms of Vg1-like UV-binding proteins either sensitive or resistant to the metal chelating agent 1, 10-phenanthroline (OP) were identified in the extracts of zebrafish early embryos. The binding of
weak acidic proteins in OP-sensitive fractions to (6-4) -photoproducts (6-4PPs) was significantly inhibited by metal chelation, and this inhibition correlated well with the instability of the 35 kDa Vg1-like polypeptide. In contrast, 6-4PP-specific binding produced by proteins having pIs about 7 to 8 in OPresistant fractions was resistant to and often stimulated by metal chelation.
Four 25 kDa Vg1-like DNA damage-recognition polypeptides had been isolated from an OP-stimulated fraction.
Using a supercoiled plasmid as a DNA repair substrate, the extracts of zebrafish embryos were able to induce a damage-specific DNA incision in the presence of ATP, generating a UV-dependent increase in the level of openform plasmid. As the level of UV-independent DNA incision was increased by
the presence of an anti-Vg1 antibody, Vg1-like UV-binding proteins might be the damage-recognition factors in embryonic NER. A repair system composed of multiple proteins in zebrafish extracts was believed to induce the UVdependent DNA incision, since each protein fraction obtained after isoelectrofocusing showed a much weaker cutting effect than the crude extracts.

總目次 1
中文摘要 3
英文摘要 7
名詞縮寫 7
壹、前言 9
一、紫外光傷害DNA 之辨識因子 9
A. Photolyases 9
B. High mobility group (HMG) 10
C. UV-damaged DNA-binding protein (UV-DDB) 10
D. TATA-box binding protein (TBP) 11
二、核酸切割修補 (NER) 辨識蛋白 11
貳、實驗材料與方法 12
一、材料 12
二、實驗方法 18
A. 斑馬魚胚胎之收集及萃取液之製備 18
B. 蛋白質濃度測定 19
C. 製備級蛋白質等電點分離試驗 19
D. 紫外光照射DNA 受質之處理 25
E. 切割試驗 25
參、結果 28
一、以凝膠阻抗實驗偵測斑馬魚胚胎發育時期DNA 損害辨識蛋白 28
二、金屬螯合劑1,10 -phenanthroline (OP) 及鹽離子影響12 hr 胚胎萃取液之辨識能力 28
三、西方墨點法實驗偵測辨識紫外線傷害DNA 之類似Vg1 蛋白 29
四、斑馬魚胚胎中UV 辨識蛋白之可能功能性鑑定 30
五、抗體對斑馬魚胚胎萃取液中切割UV-damaged DNA 能力之影響 30
六.、斑馬魚12 小時萃取蛋白辨識紫外線傷害DNA 辨識形式 30
肆、討論 31
伍、參考文獻 36
陸、附圖 41

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