Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90088
標題: Biochemical Characterization of RuvB-like DNA Helicases in Arabidopsis thaliana
阿拉伯芥 RuvB-like 核酸解旋酶之生化特性鑑定
作者: 蕭崇仁
Chung-Jen Hsiao
關鍵字: 阿拉伯芥
核酸解旋酶
Arabidopsis thaliana
RuvBL
DNA Helicase
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摘要: RuvBL1/RuvBL2 (RuvB-like) 為兩種ATP依賴型的核酸解旋酶,在真核生物中從酵母菌到人類均具有高度保留性,為一不可或缺的蛋白。廣泛出現於多種蛋白或核酸-蛋白複合體中,參與包括DNA複製、轉錄、修復、染色體重組等活動,並與細胞癌化過程高度相關,人類的RuvB-like核酸解旋酶因而被廣泛研究,但植物界的同功酵素則極少有文獻探討。我們透過生化特性鑑定,探討其在植物體內的生物功能。與人類或酵母菌不同的是,阿拉伯芥具有一個RuvBL1和兩個RuvBL2,我們分別將之命名為RuvBL1、RuvBL2-3與RuvBL2-5。兩個RuvBL2之間胺基酸序列具有91%相似度,但僅RuvBL2-5與RuvBL1轉錄時空相似,均以成熟種子中表現量最高,懷疑兩者也許可形成蛋白複合體。為進行酵素活性分析,以RT-PCR取得全長的cDNA後,於大腸桿菌表現野生型與無酵素活性(catalytically inactive)的Nus-tag及SUMO-tag融合蛋白,以Ni-NTA 樹脂純化。利用兔子製備免疫血清,取得RuvBL1或RuvBL2專一的抗體,經西方點墨法確認抗體具有專一性。然而,檢視ATPase活性的結果發現,切開Nus-tag或SUMO-tag之重組蛋白活性均極低,亦無DNA helicase活性,且其ATPase活性均不受酵素用量、協同酵素存在、或是DNA cosubstrate的提升。未切開Nus-tag或SUMO-tag之重組蛋白活性均稍高,但仍待重複實驗確認。推測酵素裁切不利於保持重組蛋白活性,後嘗試構築僅6His-tag融合之重組蛋白,但質體構築不順利,尚未取得所有質體,故無法進行後續實驗。嘗試於菸草暫時性表現RuvBLs,希望以免疫共沉澱法觀察蛋白間的互動關係,結果發現暫時性表現之蛋白質無法水溶或無表現,故無法進行共免疫沉澱分析。
RuvBL1/RuvBL2 (RuvB-like) proteins are two ATP-dependent DNA helicase highly conserved in eukaryote from yeast to human. They present in various protein and nucleoprotein complexes which play roles in cellular processes including replication, transcription, chromatin remodeling, DNA repair, etc. Acting as indispensable enzymes and playing critical roles in major pathways closely linked to cancer, RuvBLs were heavily studied in human but much less in plant. We aim to characterize their roles firstly via biochemical analysis. Unlike yeast and human, Arabidopsis thaliana possesses one RuvbL1 but two RuvBL2 genes, named as RuvBL1, RuvBL2-3 and RuvBL2-5, with the latter two encode proteins sharing 91% sequence similarity with each other. As RuvBL1 share similar expression profile with RuvBL2-5 but not RuvBL2-3, we speculate that the former two may form complexes for cooperative responses within cells. Full-length cDNA clones were obtained by RT-PCR and confirmed by DNA sequencing. To analyze enzyme activities of RuvBLs, we expressed wild type and catalytically inactive (CI) forms of Nus-RuvBLs or SUMO-RuvBLs in E. coli and purified them by Ni-NTA resin. Antiserum against RuvBL1 and RuvBL2-5 were generated and confirmed for their specificities. Nevertheless, activities of ATPase/helicase from cleaved Nus-RuvBLs and SUMO-RuvBLs were barely detectable, no matter more enzymes, partner enzymes, or different cosubstrates were employed. Suspected ATPase activities were found only from the uncleaved Nus-RuvBLs and SUMO-RuvBLs, however, require confirmation again. Recombinant RuvBLs which omit the large fusion tags together with co-expression of RuvBL1/2 will be attempted in various E. coli strains, hoping to produce proteins with enzyme activities. Moreover, to examine interactions between RuvBLs, c-myc or HA- tag were fused to the N- or C- termi of RuvBL1, 2-3 and 2-5, respectively. By Agroinfiltration of Nicotiana benthamiana, however, none or just insoluble proteins were detected for most constructs. Co-expression of RuvBL1/2 in tobacco cells will be used for detection of the protein complexes by co-immunoprecipitation.
URI: http://hdl.handle.net/11455/90088
文章公開時間: 2015-08-17
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