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標題: 細菌第IIA型 DNA 拓撲異構酵素 C 端區域之晶體結構研究
Crystallographic Studies of the C-terminal Domains of the Bacterial Type IIA DNA Topoisomerases
作者: 謝東儒
Hsieh, Tung-Ju
關鍵字: Type IIA DNA topoisomerases: DNA TopoIIAs
第IIA型DNA 拓撲異構酵素
出版社: 生物化學研究所
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摘要: 第IIA型DNA拓撲異構酶(TopoIIAs)可催化暫時性的雙股DNA缺口產生,同時捕捉另一雙股DNA並促使其通過此缺口,從而改變DNA之拓撲構型。此酵素是維繫生命現象不可或缺的一種酵素,其活性可幫助生物體解決在DNA代謝過程中因其DNA雙股螺旋結構所衍生之DNA構型問題。大部分的細菌皆具有兩種高度同源但功能截然不同的第IIA型酵素:DNA gyrase與topoisomerase IV (TopoIV)。Gyrase可利用其主動引入DNA負超螺旋的活性來協助DNA複製及轉錄過程中起始與延長作用的進行。而TopoIV則偏好DNA 正向超螺旋的鬆弛作用(relaxation)與DNA解纏作用(DNA decatenation),主要負責將DNA複製完成後相互纏繞的子染色體解開。許多生化實驗證據強烈顯示,此兩種IIA型酵素之所以具有不同的活性與其C端domain (CTD)密切相關。然而CTD的結構如何致使二者具有不同功能則有待更多結構與生化之分析。 藉由X-光蛋白質晶體繞射分析,本論文已成功解析數個TopoIIAs CTD結構,包含噬熱性枯草桿菌TopoIV CTD (BsParC-CTD)以及大腸桿菌與十字花科黑腐病菌DNA gyrase CTD (EcGyrA-CTD 與XcGyrA-CTD);分析顯示此類型蛋白皆具有一個類似但卻不同於β-propeller的摺疊方式,稱為β-pinwheel摺疊。本論文首先報告在CTD結構上所觀察到的特徵,進而利用生化方法探討這些結構特徵對於酵素功能的影響與重要性。目前已證實一個高度保留且位於β-strand上的脯胺酸(proline) 能夠影響gyrase主動引入DNA負超螺旋的效率。此脯胺酸能扭曲GyrA-CTD的結構,導致帶有正電荷之GyrA-box motif在空間上的位移,促使其所屬的DNA結合區呈現非平面的扭曲;此特性與gyrase擁有較強的DNA纏繞及引入DNA負超螺旋能力相關。由於缺少GyrA-box motif,TopoIV CTD的DNA結合區則無此非平面的扭曲。此結構上的差異可解釋DNA gyrase 與TopoIV在生化活性上的特異性。而在β-sheet特定的位點引入脯胺酸可造成蛋白功能改變的觀念更可以應用於蛋白質工程技術。
Type IIA DNA topoisomerases (TopoIIAs) are essential and ubiquitous enzymes that catalyze ATP-dependent passage of one DNA duplex (T-segment) through a transient double-stranded breakage in another (G-segment). Such an activity alters DNA topology, allowing these enzymes to solve topological conflicts arising during cellular DNA transactions. Most bacteria harbor two closely related yet functionally distinct TopoIIAs namely DNA gyrase and TopoIV. DNA gyrase supports replication and transcription with its unique supercoiling activity, whereas TopoIV preferentially relaxes (+) supercoils and is the main decatenating enzyme required for chromosome segregation. Mounting biochemical evidences suggest that the specialized activities of DNA gyrase and Topo IV depend on their respective C-terminal domains (CTDs). However, the underlying structural mechanism remains enigmatic. Using X-ray crystallography, we have determined crystal structures of TopoIV CTD from B. stearothermophilus (BsParC-CTD) as well as the DNA gyrase CTDs from Escherichia coli (EcGyrA-CTD) and Xanthomonas campestris (XcGyrA-CTD). All of them adopt a β-propeller-like fold termed β-pinwheel. Through structural and biochemical analyses, several unique structural features of these domains have been recognized and tested for the functional relevance. In particular, we have identified a highly conserved β-strand-bearing proline located in GyrA-CTD which is crucial for gyrase to exhibit efficient (-) supercoiling activity. This proline introduces structural-twist in GyrA-CTD, which results in spatial lift of the basic GyrA-box motif and an overall non-planar DNA-binding surface suitable for unidirectional DNA wrapping. In contrast, due to the lack of GyrA-box motif, the DNA-binding surface of ParC-CTD is relatively flat. This structural difference may represent a key determinant for the functional distinction between the two bacterial TopoIIAs
其他識別: U0005-1806200914323300
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