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系統識別號 U0026-0812200915223217
論文名稱(中文) 篩選與高度磷化RPA2 交互作用之蛋白及其研究
論文名稱(英文) Screening and studying of the proteins interacting with hyperphosphorylated RPA2
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 鄭凱峻
研究生(英文) Kai-chun Cheng
電子信箱 s1696404@mail.ncku.edu.tw
學號 s1696404
學位類別 碩士
語文別 中文
論文頁數 111頁
口試委員 口試委員-賴明德
口試委員-鄭宏祺
指導教授-張敏政
口試委員-張明熙
中文關鍵字 交互作用 
英文關鍵字 interaction  p53  L5-L11-L23-MDM2  RPA2 
學科別分類
中文摘要 Replication protein A(RPA) 是由三個次單元蛋白RPA1 (70 kDa)、RPA2 (32 kDa) 與RPA3 (14 kDa)所組成,在生物體中RPA扮演的角色為單股DNA結合因子(single-strand DNA-binding factor)。RPA參與DNA生理上的三種程序,分別是DNA複製(replication)、DNA重組(recombination)及DNA修復(repair)。其中DNA複製及修復之間的轉換主要是藉由RPA2的磷酸化與否來進行調控。在先前的研究中發現,受到基因毒性壓力(genotoxic stress)過程,RPA2的N端會有九個位點被磷酸化之現象,但目前並不清楚高度磷酸化RPA2在生理中所扮演的角色。在實驗室先前發現去除C端之RPA2會與核糖蛋白L5有交互作用,由pull down及共同免疫沉澱發現核糖蛋白L5會與高度磷酸化RPA2有交互作用。利用site-directed mutagenesis建立模擬高度磷酸化RPA2,由模擬高度磷酸化RPA2進行共同免疫沉澱(Co-IP)、酵母菌雜交接合系統(yeast two-hybrid system)及哺乳類雜交結合系統(mammalian two hybrid system)皆證實高度磷酸化RPA2與L5有交互作用。由相關文獻推測高度磷酸化RPA2與L5之交互作用可能涉及L5-L11-L23-MDM2複合物,實驗室先前發現高度磷酸化RPA2與L11有交互作用,在共同免疫沉澱(Co-IP)實驗指出模擬高度磷酸化RPA2不只與L5有交互作用,L11也參與再其中。進一步由共同免疫沉澱(Co-IP)與酵母菌雜交接合系統(yeast two-hybrid system)中發現模擬高度磷酸化RPA2可能與L5-L11-L23-MDM2複合物有交互作用,其中L5、L23及MDM2為直接(direct)交互作用,L11為間接(indirect)交互作用。高度磷酸化RPA2與L5-L11-L23-MDM2複合物交互作用之生理意義可能是影響p53活性,由p53 response element assay及p21 promoter assay中發現knockdown RPA2及L5時p53活性可能受到影響。未來需更進一步研究高度磷酸化RPA2與L5-L11-L23-MDM2複合物交互作用影響p53之生理意義。另外,因基因毒性壓力形成高度磷酸化RPA2可在細胞內維持長時間,在這段時間內,高度磷酸化RPA2可能會與細胞內許多蛋白進行交互作用,利用酵母雜交接合系統去找尋與高度磷酸化RPA2交互作用之蛋白,推敲高度磷酸化RPA2在細胞內所扮演的角色。利用site-directed mutagenesis之方式,建立模擬高度磷酸化RPA2蛋白。利用酵母雜交接合系統由人類乳腺基因庫(Human Mammary Gland Matchmaker cDNA Library)找尋與模擬高度磷酸化RPA2交互作用之蛋白,由四百個菌落中發現有三十二個候選基因,並挑選出七個有興趣的候選基因分別為Triad3A、TLR5、TC-1、FOS、RPLP0、RPL3、FYN。這些候選基因須進一步確認與高度磷酸化RPA2的交互作用,並找尋交互作用時機與生理意義。
英文摘要 Replication protein A (RPA) is a heterotrimeric single-strand DNA-binding factor which is composed of three subunits, RPA1 (70 kDa), RPA2 (32 kDa) and RPA3 (14 kDa). RPA is critical for the “three Rs” of eukaryotic DNA enzymology: DNA replication, DNA recombination and DNA repair. The activity of human RPA in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are phosphorylated in vivo under genotoxic stress. However, the significance of RPA phosphorylation is still unclear. In our previous studies, a ribosomal protein, L5, was identified as a specific hyperphosphroylated RPA2-interacting protein. In this study, we constructed a mimicking hyperphosphorylated form of RPA2 (RPA2-7D2E) and a mimicking hypophosphorylated form of RPA2 (RPA2-9A) by site-directed mutagenesis, respectively, and used them to demonstrate that L5 could interact with the RPA2-7D2E but not RPA2-9A and RPA2 by co-immunoprecipitation (co-IP), yeast two-hybrid system and mammalian two-hybrid system. Although, our study initially demonstrated another ribosomal protein, L11, could interact with hyperphosphroylated RPA2 by using co-IP. Nevertheless, the results of yeast two-hybrid system revealed that L11 couldn’t interact with the RPA2-7D2E, suggesting that L11 might indirectly interact with hyperphosphroylated RPA2. Several studies reported that MDM2, L5, L11 and L23 could form a complex which functions to inhibit MDM2-mediated p53 ubiquitination and thus activate p53. Since our yeast two-hybrid assays indicated that L5, L23 and MDM2 could interact with RPA2-7D2E and the results of p53 response element assay and p21 promoter assay revealed that p53 activation wasn’t up-regulated in either RPA2 or L5 knockdown cells after UV irradiation. We propose that under stress conditions, p53 activation might be resulted from the hyperphosphroylated RPA2 interacting with L5, L23, and MDM2. This hypothesis needs to be further investigated. To elucidate the biological roles of the hyperphosphroylated RPA2, we also use RPA2-7D2E to screen proteins interacting with mimicking hyperphosphorylated form of RPA2 in yeast two-hybrid system. Over 400 clones were isolated and among these positive clones, 32 clones whose plasmids containing over 1000 bp length of the insert DNA fragment were selected. Several proteins were identified as the specific interactors for RPA2-7D2E. We expect to uncover the biological roles of hyperphosphorylated RPA2 through characterization of our selected hyperphosphorylated RPA2-interacting protein.
論文目次 中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 VIII
縮寫表 XI
緒論 1
1-1. RPA 1
1-2. RPA之功能 2
1-3. RPA之三個次單元體各自功能 3
1-4. RPA之磷酸化作用 4
2-1. RPL5簡介 6
2-2. 與L5交互作用之蛋白 7
研究動機 9
材料與方法 11
1. 實驗菌株 11
2. 載體 11
3. 培養基配方 12
4. 質體建構 14
5. 聚合酶連鎖反應 15
6. 構築 PCR 片段於質體中 15
7. 形質轉移 16
8. 小量質體製備 18
9. 蛋白質分析 20
10. SDS-PAGE 蛋白質電泳 22
11. 西方墨點法 23
12. 細胞培養方法 25
13. 細胞轉染 29
14. 哺乳類雜交接合(mammalian two hybrid)系統 29
15. 共同免疫沉澱法(Co-immunoprecipitation) 30
實驗結果 32
1. 高度磷酸化RPA2與L5之交互作用 32
1-1. 以內源性RPA2進行共同免疫沉澱(Co-immunoprecipitation) 32
1-2. 模擬高度磷酸化RPA2之基因片段 32
1-3. 酵母菌雜交接合系統(Yeast two-hybrid system) 33
1-4. 以模擬高度磷酸化RPA2進行共同免疫沉澱 34
1-5. 哺乳類細胞雜交接合系統(Mammalian two-hybrid system) 34
2. 高度磷酸化RPA2與L5-L11-L23-MDM2之交互作用 35
2-1. 高度磷酸化RPA2與L11之交互作用 36
2-2. 高度磷酸化RPA2與L5-L11-L23-MDM2之交互作用 36
3. 高度磷酸化RPA2與L5-L11-L23-MDM2交互作用之生理意義 38
3-1. 高度磷酸化RPA2與L5-L11-L23-MDM2交互作用影響p53之活化 38
3-2. p53活化影響下游基因p21 39
4. 利用Yeast two-hybrid找尋可與高度磷酸化RPA2交互作用之蛋白 40
4-1. pGBKT7-RPA2之建立 40
4-2. bait gene的評估 40
4-3. 由基因庫找尋與高度磷酸化RPA2交互作用的蛋白 41
5. 模擬高度磷酸化RPA2或RPA2與候選基因之交互作用 41
5-1. 模擬高度磷酸化RPA2與候選基因之交互作用 41
5-2. RPA2與候選基因之交互作用 42
5-3. 利用共同免疫沉澱法確認模擬高度磷酸化RPA2與候選基因之交互作用 44
實驗討論 45
1. 高度磷酸化RPA2與L5之交互作用 45
2. 模擬高度磷酸化RPA2之蛋白 45
3. 高度磷酸化RPA2與L5-L11-L23-MDM2之交互作用 46
4. 高度磷酸化RPA與L5-L11-L23-MDM2交互作用影響p53之假說 47
5. 高度磷酸化RPA2與L5-L11-L23-MDM2交互作用之生理意義 49
6. 利用Yeast two-hybrid找尋可與高度磷酸化RPA2交互作用之蛋白 50
參考文獻 52
實驗結果圖表 59
附錄 102
自述 111
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