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研究生:陳意淳
研究生(外文):Yi-Chun Chen
論文名稱:EB病毒之蛋白質激酶BGLF4對細胞內DNA修復能力的影響
論文名稱(外文):The Effect of Epstein-Barr Virus Protein Kinase BGLF4 on Cellular DNA Repair Activity
指導教授:陳美如陳美如引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:58
中文關鍵詞:EB病毒蛋白質激酶BGLF4
外文關鍵詞:EBV protein kinase BGLF4
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細胞內的蛋白質激酶在訊息傳導、細胞週期的調控等弁鄐W扮演著重要的角色,而病毒蛋白質激酶除了可以調節病毒的複製週期外,也可能透過磷酸化宿主蛋白質,影響細胞蛋白質的弁遄CEB病毒的BGLF4是EB病毒唯一已知的Ser/Thr蛋白質激酶(protein kinase),屬於溶解期早期表現的基因產物,BGLF4有自我磷酸化的現象,並有磷酸化EA-D、EF-1δ和ganciclovir的能力。在本篇研究中,以酵母菌雙雜交(yeast two hybrid)的方式篩選帶有EB病毒的類淋巴球母細胞株互補基因庫,發現BGLF4可分別與基因體核苷酸切除修復中負責辨認錯誤DNA的蛋白質XPC (xeroderma pigmentosum complementation group C),和腫瘤抑制因子BRCA1的共同抑制因子ZBRK1 (Zinc finger and BRCA1-interacting protein with a KRAB domain 1) 產生交互作用。因為這兩個蛋白質都參與細胞內DNA修復機制,故進一步探討BGLF4與XPC之間可能的交互作用,同時也研究BGLF4對細胞DNA修復能力可能的影響。首先利用共同免疫沉澱法證實無激酶活性的BGLF4突變蛋白質和HA tagged XPC蛋白質片段的交互作用。以免疫螢光染色觀察BGLF4與XPC在HeLa細胞內表現的位置,發現兩蛋白質在細胞內位置有部分重疊。在宿主細胞質體再活化試驗中,發現BGLF4可以促進p53缺失的H1299/bcl2細胞修復受損DNA的能力,而在XPC突變的XP4PA-SV細胞中則無此現象。至於BGLF4促進細胞修復DNA的能力是否經由需要XPC的機制,或是藉由其他的途徑,仍需進一步的研究。
Cellular protein kinases are known to be involved in regulating many cellular events such as signal transduction or cell cycle progression. Viral protein kinases are expected to play important roles in modulating not only viral replication machinery but also the biological function of infected cells. BGLF4, which expresses as an Epstein-Barr virus (EBV) early gene, is the only identified Ser/Thr protein kinase of EBV. The kinase activities of BGLF4 were demonstrated for autophosphorylation and phosphorylating viral DNA polymerase accessory factor EA-D, cellular translation elongation factor EF-1δ and ganciclovir. To identify possible cellular substrates of BGLF4, yeast two-hybrid system was employed to screen a cDNA library derived from an EBV positive lymphoblastoid cell line in this study. Two cellular proteins identified were global genome nucleotide excision repair initiator XPC (xeroderma pigmentosum complementation group C) and ZBRK1 (Zinc finger and BRCA1-interacting protein with a KRAB domain 1, which was implicated as a corepressor of BRCA1). Since both proteins are involved in the DNA repair system, the interaction between BGLF4 and XPC, and the possible effect of BGLF4 on cellular DNA repair machinery were further analyzed. The interaction between a kinase dead BGLF4 mutant and an HA tagged XPC truncated protein were confirmed by co-immuno- precipitation with reciprocal antibodies. BGLF4 and XPC were found partially colocalized in HeLa cells as demonstrated by immunofluorescence. In host cell reactivation assay, BGLF4 was found to enhance the DNA repair activity in H1299/bcl2 cell that harbors defective p53, but not in XPC mutanted XP4PA-SV cell. Whether BGLF4 enhances the DNA repair activity through an XPC dependent mechanism or other pathway needs to be further investigated.
中文摘要 -Ⅰ-
英文摘要 -Ⅱ-
前言 01
材料與方法 14
結果 23
討論 28
圖表 33
參考文獻 50
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