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研究生:張華旗
研究生(外文):Hua-Ci Jhang
論文名稱:蛋白質與核酸間交互作用之親和性與專一性設計
論文名稱(外文):Redesign of Protein-DNA Interactions in Binding Affinity and Specificity
指導教授:翟建富翟建富引用關係袁小琀
指導教授(外文):Kin-Fu ChakHanna S. Yuan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
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:73
中文關鍵詞:螢光共振能量轉移計算方式鹼基偏好性
外文關鍵詞:Fluorescence Resonance Energy Transfercomputational methodsbase preference
相關次數:
  • 被引用被引用:1
  • 點閱點閱:64
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
蛋白質與DNA 間的辨識現象在許多生理現象中扮演著中心的色,
其範圍的涵蓋從DNA 的複製、轉錄、重組和修復甚至到染色體的凝聚和細胞凋亡中的DNA 裂解,因此廣泛的被視為研究的題材。其重要性也在於藉由這些研究將有助於闡明支配蛋白質與DNA 間結合選擇性的基本原則。在本論文中,我們利用E7大腸桿菌素作為分子模擬的材料
,進一步探討蛋白質與DNA 間的交互作用情形。
E7大腸桿菌素本身具有非專一性的內切酶活性,可將目標細胞內DNA或RNA裂解。為了保護宿主細胞免受該毒性的危害,宿主細胞會同時表現一個E7免疫蛋白質,用來抑制E7大腸桿菌素的核酸水解酶活性
。目前,E7大腸桿菌素的毒性區塊與一個具有8 個鹼基對雙股DNA 之複合物晶體結構已解出,根據該結構選擇了三個牽涉到與免疫蛋白質結合的氨基酸作突變,按此分析,這些突變種蛋白,包含了R520A、K525A、K537A、R520A+K537A 及K525A+K537A 應該在DNA 的結合上呈現較低的親和力。然而,令我們訝異的是,這些突變種蛋白不論是在plasmid nicking 實驗或是利用FRET(螢光共振能量轉移)方法所量測到的核酸水解酶活性,均與原生種蛋白近似。
我們也利用計算的方式進行nuclease-ColE7突變種蛋白的設計,
欲藉此設計得到具有高的DNA 結合力以及具有不同序列專一性的蛋白
。根據計算結果,我們建構了數個nuclease-ColE7 突變種蛋白,包含了K490R、D493N、D493Q 與K497R,並發現D493N、D493Q 與K497R 具有增加的DNA 結合能力,在cleavage site mapping 實驗中,亦可
觀察到D493Q 突變種蛋白與原生種蛋白相較之下有不同的鹼基偏好。從這些結果可知,利用結構的資訊並配合理論計算的方法,對產生一個具備不同DNA 結合能力及鹼基偏好性的內切酶而言,確實為一種有用的策略。
Recognition between proteins and DNA has been studying extensively because it plays the central role for all kinds of biological events, from DNA replication,transcription, recombination and repair to chromosome assembly and DNA degradation in apoptosis. It is therefore of crucial
importance to elucidate the underlying principles that govern the binding selectivity between proteins and DNA. Here we use ColE7 as a model system to study the interactions between a protein and DNA molecules.
ColE7 contains non-specific endonuclease activity that it digests cellular DNA or RNA in target cells. In order to protect the host cell from the cytotoxic nuclease activity of ColE7, an immunity protein, Im7, is co-expressed with ColE7 to inhibit the nuclease activity of ColE7. The crystal
structure of the nuclease domain of ColE7 (nuclease-ColE7)
in complex with an 8-bp duplex DNA has been determined recently at 2.5 Å resolution.Based on the crystal structure
of nuclease-ColE7/DNA complex,three key residues involved both in binding of Im7 and DNA were selected for mutations.
These mutants, R520A, K525A, K537A, R520A+K537A and
K525A+K537A, should have lower affinity for DNA binding. However, to our surprise, the nuclease activity of these mutants were similar to that of wild-type enzyme as tested by plasmid nicking assays and FRET (Fluorescence Resonance Energy Transfer) methods.
We then used computational methods to design mutated nuclease-ColE7 proteinswith higher DNA-binding affinity and different sequence specificity. Several nuclease-ColE7 mutants,K490R, D493N, D493Q and K497R, were constructed based on computational redesign. We found that D493N, D493Q and K497R had increased DNA-binding affinity. The cleavage site mapping assay further showed that D493Q digested DNA with a different base preference as compared to the wild-type enzyme.
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