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標題: | 嗜熱菌拓樸異構酶VI結合去氧核醣核酸之複合體結構解析 Toward Structural Analysis of Topoisomerase VI from the Thermophilic Archaea Nanoarcharum equitans in Complex with DNA |
作者: | Wen-Yang Kuo 郭文揚 |
指導教授: | 詹迺立 |
關鍵字: | 拓樸異構?VI,拓樸結構,第二型拓樸異構?,X射線繞射結晶學,嗜熱菌, topoisomerase VI,DNA topology problem,type II topoisomerase,X-ray crystallography,thermophile, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | DNA拓樸異構酶為一類生物體中不可或缺的酵素,負責解決DNA在進行複製、轉錄、重組、修復等過程中所形成的拓樸結構問題,像是複製過程中產生的DNA雙股連鎖,或是在轉錄的過程中因轉錄複合體推動而形成的正、負超螺旋等,這些較複雜的DNA結構若未被移除,會導致染色體的不穩定,以及細胞功能的異常。拓樸異構酶即是利用活性中心的酪胺酸,對DNA之磷酸基團進行親核性攻擊,藉由催化可逆的轉酯反應形成磷酸酪胺基鍵(phosphotyrosyl bond),造成DNA短暫的斷裂,並使得另一股DNA或是另一DNA雙股分子得以通過此缺口,因此可改變DNA的拓樸結構,之後透過第二次的轉酯作用,使DNA重新黏合。基於結構以及機制的不同,拓樸異構酶可被分為第一型與第二型兩大類,其差別在於前者只切開單股,後者則可同時切開DNA雙股,而藉由酵素的序列與催化特性,第二型酵素可再進一步分為IIA與IIB兩亞型。拓樸異構酶VI是IIB亞型的唯一成員,主要存在於古細菌及部分植物中,其於生理狀態下為異質四聚體,部分序列與IIA酵素以及參與DNA調節的蛋白具有相似性。先前的研究指出,拓樸異構酶VI的作用機制與IIA酵素類似,兩者皆需要結合與水解ATP來催化DNA雙股的斷裂及解旋,但是主要功能區域在序列以及立體結構的排列卻有顯著差異。例如,拓樸異構酶VI會產生的DNA斷口兩端相隔兩個鹼基對,但其他IIA酵素引起的斷裂則是相隔四個鹼基對。此外,參與轉酯化反應的酪胺酸於拓樸異構酶VI中出現在α螺旋上,而IIA蛋白則位在β-hairpin環上。因此,我們認為拓樸異構酶VI結合與切割DNA的機制上勢必與IIA酵素有明顯的差異,但目前為止還沒有任何關於此酵素與DNA形成之複合體的結構資訊,所以我們希望透過X射線繞射結晶學的方式,更進一步研究其結構與機制。我們成功表達並純化功能完整之嗜熱菌拓樸異構酶VI蛋白,並透過活性測試確認其對ATP及二價離子的依賴性,並得知反應的最適溫度約在70至80度之間。我們亦針對其序列偏好性設計出可能與其專一性結合的DNA受質,之後將使用此雙股DNA進行養晶條件測試。 DNA topoisomerases are essential enzymes that are ubiquitously present in all organisms for resolving the entangled or interlinked DNA structures resulted from replication, transcription, recombination, DNA repair, and chromatin remodeling. Failure in the removal of these higher-order topological DNA structures is known to cause genome instability and dysregulation of cellular functions. To alter DNA topology, DNA topoisomerases utilize a tyrosine residue located in the active site to catalyze a reversible transesterification reaction. During this process, the active site tyrosine attack the DNA phosphoribosyl backbone to form a phosphotyrosyl linkage and a concomitant breakage of the phosphodiester bond, which creates a transient DNA break to permit the passage of another DNA strand or DNA duplex through the opening. The DNA break can then be resealed through the second transesterification reaction with the regeneration of the active site tyrosine. Based on structural and mechanistic distinctions, these enzymes are classified into type I and II, which are distinguished by their ability to cleave one or both strands of DNA duplex. These two groups can be further divided into A and B subfamilies. Among them, topoisomerase VI (Topo VI) is the sole member of type IIB topoisomerase, found mainly in archaea and plants. Topo VI functions as an A2B2 heterotetramer, whose subunits share sequence homology with type IIA enzymes and other proteins involved in DNA processing. Previous studies showed that, similar to the type IIA enzymes, Topo VI also possesses an ATP-dependent duplex passage activity, but the domains of Topo VI subunit are organized differently along the polypeptide sequence and are placed in spatially distinct positions. In addition, unlike the type IIA enzymes, Topo VI produces a 2-base- rather than a 4-base-staggered DNA break. Moreover, the active site tyrosine of Topo VI is located within a helix rather than a loop region as seen in type IA and IIA enzymes. Therefore, we suspect that Topo VI may employ a distinct mechanism with respect to DNA binding and cleavage. So far, there is no direct structural information available to elucidate the interaction between Topo VI and DNA. Therefore, we attempt to characterize the structure of Topo VI in complex with a DNA duplex by X-ray crystallography. To this end, we have successfully purified and reconstituted a chimeric Topo VI holoenzyme from a thermophilic archaea. Functional analyses indicated that the Topo VI we prepared possesses an ATP- and temperature-dependent relaxation activity, with optimal activity being observed at 70~80˚C. We have initiated crystallization screens on the Topo VI-DNA binary complex. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51773 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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