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研究生:賴允婷
研究生(外文):Yeun-Tyng Lai
論文名稱:人類核苷酸切除修復因子hHR23A和hHR23B的蛋白質交互作用研究及對於蛋白質降解之調節
論文名稱(外文):The protein interaction studies of the two human nucleotide excision repair factors hHR23A and hHR23B and their roles as protein degradation regulators
指導教授:黃溫雅黃溫雅引用關係郭育良郭育良引用關係
指導教授(外文):Wenya HuangYue-Liang L. Guo
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:68
中文關鍵詞:hHR23AhHR23B酵母菌雙雜交蛋白質降解核苷酸切除修復
外文關鍵詞:hHR23AhHR23Byeast two-hybridprotein degradationNER
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DNA修復是細胞將基因上的損壞移除的重要機制,而核苷酸切除修復主要修復經紫外線照射或致癌化學藥品造成的大型DNA構形改變,若核苷酸切除修復的基因發生突變,會引發著色性乾皮症。Rad23是一出芽酵母菌的核苷酸切除修復因子,已知和蛋白質的降解也有關,在人類則有兩個同源基因:分別是hHR23A和hHR23B,但它們在核苷酸切除修復上的功能尚不清楚,只知兩者的序列極相似,在N端有一UBL功能區域,而中間和C端各有UBA1及UBA2功能區域。已知經由酵母菌雙雜交法篩選,我們發現hHR23A和hHR23B與26S proteasome的組成分子TRAP2和S5a以及另一分子ubiquitin有交互作用。
為了研究hHR23A/B是否會經由UBL和UBA功能區域產生交互作用,我們設計了許多含不同功能區域的hHR23A/B蛋白質進行酵母菌雙雜交試驗;結果證明hHR23A與hHR23B有交互作用,而且不是透過UBL功能區域,另外hHR23B則會形成同二聚物。又因為hHR23A/B和26S proteasome有關係,或許暗示它們可能參與蛋白質降解的步驟,故根據N-end rule,我們設計了包括ubiquitin、DHFRts及-galactosidase三段基因的reporter去測試蛋白質降解速度,現在正在評估它的適用性。藉由蛋白質交互作用實驗以及蛋白質降解試驗,我們可以更加瞭解hHR23A和hHR23B的性質及功能。

DNA repair is the major cellular mechanism by which the DNA damages on the genome are removed. The nucleotide excision repair (NER) mainly operates on a large spectrum of base damages, particularly that produced by ultraviolet (UV) irradiation and carcinogenic chemicals which produce bulky, helix-distorting lesions in DNA structure. Xeroderma pigmentosum (XP) syndromes are the diseases that are caused by mutations in NER genes. Rad (radiation-sensitive) 23 is the NER factor in the yeast S. cerevisiae and may regulate NER pathway by associating with protein degradation. hHR23 (human homolog of Rad23) A and hHR23B are two sequence homologs of Rad23; however, their roles in NER in vivo remain to be elucidated. hHR23A and B are highly homologous with each other in that both of them contain the ubiquitin-like (UBL) domain at the N-termini and two ubiquitin-associated (UBA) domains at the C-termini. In the yeast two-hybrid screening assay using hHR23A/B as the bait, three proteins were identified: the 26S proteasome subunits (1) TRAP2 and (2) S5a, and (3) ubiquitin.
To study the intermolecular or intramolecular interactions through the UBL and UBA domains, we designed some hHR23A/B truncated forms and used yeast two-hybrid to assay. Our data have shown that hHR23A interacts with hHR23B even without the UBL domain of hHR23B. In addition, hHR23B and hHR23B may become homodimer. Because of the interactions of hHR23A/B with the 26S proteasome, we suggested hHR23A/B may be involved in protein degradation. By N-end rule, we designed a reporter gene which contains ubiquitin, different N-end DHFRts and -galactosidase to examine the protein degradation in vivo. The preliminary results of these reporters have shown the relationship with N-end rule, now we just test the influences of protein degradation inhibitor. Through experiments of the protein interaction and the protein degradation reporter system, we will understand more about the properties and functions of hHR23A and hHR23B.

表目錄…………………………………………………………………………1
圖目錄…………………………………………………………………………2
1. 緒論……………………………………………………………………….3
1.1 前言………………………………………………………………………3
1.2 核苷酸切除修復…………………………………………………………4
1.3 hHR23A與hHR23B………………………………………………………..5
1.4 動機………………………………………………………………………7
1.5 原理………………………………………………………………………7
1.5.1 酵母菌雙雜交試驗…………………………………………………..7
1.5.2 蛋白質降解試驗……………………………………………………..8
2. 材料與方法……………………………………………………………….9
2.1 細胞株與化學藥品………………………………………………………9
2.2 酵母菌雙雜交試驗………………………………………………………9
2.2.1 Clone製備…………………………………………………………….9
2.2.2 大腸桿菌的質體轉化……………………………………………….12
2.2.3 小量質體抽取……………………………………………………….12
2.2.4 酵母菌的質體轉化………………………………………………….13
2.2.5 酵母菌蛋白質的萃取與定量……………………………………….14
2.2.6 西方墨點…………………………………………………………….14
2.2.7 beta-galactosidase濾紙試驗…………………………………….15
2.3 蛋白質降解試驗……………………………………………………….16
2.3.1 Clone製備………………………………………………………....16
2.3.2 用kit抽取質體……………………………………………………..18
2.3.3 人類細胞的質體轉染……………………………………………….18
2.3.4 反轉錄聚合酶連鎖反應…………………………………………….19
2.3.5 以冷光儀偵測beta-galactosidase……………………………….20
3. 結果………………………………………………………………………21
3.1 酵母菌雙雜交試驗…………………………………………………….21
3.1.1 以西方墨點測試送入酵母菌Y187的質體之表現………………...21
3.1.2 hHR23B與hHR23A/B有交互作用……………………………….....21
3.1.3 UBL功能區域和UBA1或UBA2功能區域沒有交互作用………......22
3.1.4 UBL、UBA1或UBA2功能區域和hHR23A/B也沒有交互作用…......22
3.1.5 hHR23A和hHR23B的交互作用不需UBL功能區域……………......23
3.1.6 hHR23A/B和單一單位的ubiquitin有交互作用…………………..24
3.2 蛋白質降解試驗……………………………………………………….24
3.2.1 以反轉錄聚合酶連鎖反應偵測送入293T細胞的reporter之表現.24
3.2.2 各個reporter的beta-galactosidase活性……………………….25
4. 討論………………………………………………………………………26
參考文獻…………………………………………………………………….29
表…………………………………………………………………………….37
圖…………………………………………………………………………….48

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