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標題: | EB病毒BKRF3尿嘧啶醣苷酶之功能及其與病毒核酸複製區蛋白質之交互作用 Characterization of Epstein - Barr virus BKRF3 Uracil-DNA Glycosylase and Its Interaction with Proteins in Viral Replication Compartment |
作者: | Shu-Ming Chang 張書銘 |
指導教授: | 陳美如(Mei-Ru Chen) |
關鍵字: | EB病毒,BKRF3,尿嘧啶醣苷,酶,UNG2,BALF5,Rta, Epstein-Barr virus,BKRF3,Uracil-DNA glycosylase,UNG2,BALF5,Rta, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 尿嘧啶醣苷酶(Uracil DNA glycosylase,UDG)是在細胞中參與鹼基移除修復(Base excision repair)機制中的首要酵素。EB病毒溶裂期早期基因BKRF3的蛋白質產物為一尿嘧啶醣苷酶,在siRNA及尿嘧啶醣苷酶抑制物實驗中發現,BKRF3與宿主細胞尿嘧啶醣苷酶UNG2 (Uracil-N-Glycosylase 2) 對於EB病毒核酸複製效率是重要的。本研究希望進一步探討BKRF3功能,首先以大腸桿菌系統證明BKRF3與UNG2類似,具有G:U配對修復之能力。接著以BKRF3可提引細胞株,觀察BKRF3對細胞之影響。過度表現UNG2會引起DNA損傷訊息活化或減緩細胞生長速度,但BKRF3則無法明顯增加整體酵素活性而減緩細胞生長速度。為進一步了解BKRF3在病毒核酸複製所扮演的角色,觀察在溶裂期進行時UNG2及BKRF3表現量在EREV8細胞中變化,發現UNG2表現量在溶裂期晚期下降,此時而BKRF3表現量增加,因此可能具補足逐漸下降的UNG2功能,在溶裂期後期維持病毒DNA複製效率。以免疫螢光染色法及細胞質核分離萃取進一步發現當BKRF3單獨表現時,主要侷限於細胞質中,在溶裂期時,BKRF3聚集至細胞核的病毒核酸複製區。BKRF3可與DNA合成酶BALF5及Rta共同免疫沉澱,BKRF3在溶裂期可能必須透過與病毒核酸複製區蛋白質交互作用,增加其入核效率。而以共同轉染方式發現單獨表現DNA合成酶BALF5及轉活化因子Rta無法增加BKRF3入核效率,可能需要其他病毒蛋白質形成完整之病毒核酸複製複合體才能有效率使BKRF3入核。另以GST pull-down法,觀察BKRF3與DNA合成酶BALF5及轉活化因子Rta產生交互作用區域,發現BKRF3利用其C端與DNA合成酶BALF5及轉活化因子Rta交互作用。經由上述實驗結果,推測BKRF3透過蛋白質之間交互作用,參與在病毒DNA複製過程中以增進DNA複製的效率及正確性。 Uracil DNA glycosylase (UDG) is the primary enzyme that participates in the DNA base excision repair (BER) mechanism. BKRF3 of Epstein-Barr virus (EBV) encodes an UDG enzyme that is expressesed in lytic stage. Both BKRF3 and the cellular major UDG (Uracil-N-Glycosylase 2, UNG2) contribute to viral lytic DNA replication as revealed by siRNA experiments and a bacteria phage inhibitor PB2. This study is aimed to further characterize the biologic function of BKRF3. Firstly, similar to mammalian UNG2, BKRF3 is able to repair G:U mismatch in E. coli. A tet-on inducible system in RHEK293 cells is adapted to establish BKRF3 inducible cells for studying its intracellular effects. Different from cellular UNG2, expression of BKRF3 neither enhances total UDG activity significantly nor induces DNA damage or cell growth retardation. Analysis of the expression pattern of cellular UNG2 and BKRF3 in EBV replicating EREV8 cells reveals that the expression of BKRF3 sustains through viral replication, whereas UNG2 decreases at the late stage of viral replication, suggesting BKRF3 may compensate the UDG activity at late stage of virus replication. Immunofluorescence staining and subcellular fractionation further demonstrate that BKRF3 mainly distributes in the cytoplasm when it is expressed alone, but it is recruited to viral DNA replication compartment in the nucleus of EREV8 cells with lytic viral DNA replication. BKRF3 is co-immunoprecipitated with viral DNA polymerase BALF5 and the immediate early transactivator Rta from EREV8 cells. However, transient expression of BKRF3 with BALF5 or Rta is not sufficient to enhance the nuclear retention of BKRF3, suggesting other viral proteins or viral DNA dependent mechanisms are involved in recruiting BKRF3 to viral DNA replication compartment. GST-pull down assay indicates that the carboxyl half of BKRF3 contributes to its interaction with BALF5 and Rta. Taken together, data here suggest that BKRF3 participates in the viral DNA replication machinery through direct protein-protein interaction, which may ensure the fidelity and efficiency of viral DNA replication. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26608 |
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