- 學位論文
大腸桿菌旋轉酶與DNA交互作用之結構解析
Structural Analysis of DNA-Binding by Escherichia coli Gyrase
摘要
DNA拓樸異構酶可藉由其切割—接合DNA的活性造成DNA拓樸結構的改變，進而影響DNA的生理活性。在四大類拓樸異構酶家族中，Type IIA拓樸異構酶廣泛的分布於細菌與真核生物界。本型酵素可暫時性的切斷雙股DNA，並利用ATP結合和水解所引發的蛋白構形變化來驅動另一雙股DNA通過斷裂處，以因應生物體所面臨的DNA拓樸問題。 所有細菌都含有DNA gyrase (DNA旋轉酶)此種功能特異化的Type IIA酵素，它可藉由ATP水解所提供的能量在DNA中引入負超螺旋，gyrase也是目前已知唯一具有此功能的拓樸異構酶。此特殊活性使得gyrase能有效的移除複製叉和RNA聚合酶前方的DNA正超螺旋，並可使細菌基因體處於適度的負超螺旋態。由於gyrase在細菌中扮演不可或缺的角色，所以它是良好的藥物標的蛋白，如fluoroquinolones類型的抗生素可嵌進gyrase誘發的DNA斷裂處，抑制其酵素活性並引發DNA損傷，達成抑菌的效果。 文獻指出gyrase功能的特異性，主要取決於其GyrA subunit上之C-terminal domain (CTD) 能以右旋方式纏繞DNA，形成一個正向的DNA交叉，再利用Type IIA酵素典型的催化機制將此交叉翻轉為負超螺旋，去除CTD的gyrase會喪失引入負超螺旋的活性。雖然gyrase CTD與DNA-binding and cleavage core (DBCC)之晶體結構以及gyrase引入負超螺旋的可能模式已於近年陸續發表，但目前對於其催化反應的詳細動態過程依舊不清楚，所以本研究希望以蛋白質結晶學技術，試著培養以融合蛋白型式表達包含CTD與DBCC二者之融合蛋白、並解析其與DNA與fluoroquinolones形成之三重複合體的晶體結構，以嘗試解答CTD如何與DNA交互作用並與DBCC搭配，以執行其引入負超螺旋的活性。目前已成功得到大量高品質且具有DNA結合與切割活性的融合蛋白，並已展開三重複合體的晶體培養。
並列摘要
DNA topoisomerases are essential enzymes that are responsible for modulating the topological states of cellular DNA. Among the four classes of topoisomerases, the type IIA family is widely distributed in bacteria and eukaryotes and is involved in several types of topological transformations, such as catenation and decatenation of DNA rings, relaxation of supercoiled DNA, and introduction of negative DNA supercoils. The catalytic cycle of type IIA enzyme includes transient cleavage of a double-stranded DNA and passage of another duplex DNA through followed by relegation of this break. DNA gyrase is a functionally specialized bacterial type IIA topoisomerase with an unique ATP-dependent negative supercoiling activity. As the only enzyme capable of introducing negative DNA supercoils, gyrase is particularly effective in removing the positive DNA supercoils that are accumulated in front of the replication fork or advancing RNA polymerases. In addition, gyrase keeps the bacterial genome in a slightly negative supercoiled state to facilitate various DNA transactions. Because of its vital roles in bacteria, gyrase has served as a primary target of antibacterial drugs. For example, the fluoroquinlone compounds are highly successful antibiotics which can induce DNA damage in bacteria and thus their death by stabilizing the gyrase-mediated DNA breaks. Previous studies revealed that the unique negative supercoiling activity of gyrase is dependent on the C-terminal domain of the GyrA subunit (GyrA-CTD), which can wrap a duplex DNA segment into a positive crossover to facilitate its subsequent conversion into a negative DNA superhelical turn. Removal of GyrA-CTD abolishes gyrase’s activity to introduce negative supercoils. Therefore, GyrA-CTD is required for the unique supercoiling activity of gyrase. To elucidate the structural basis of gyrase-catalyzed negative supercoiling reaction, it is important to understand how a piece of DNA is bound and shaped by the enzyme. Although the crystal structure of GyrA-CTD and gyrase DNA-binding and cleavage core (DBCC) has been determined, no information is available regarding how the functional coordination is achieved between this domain and the enzyme’s core catalytic module. Therefore, we would like to address this question by determining the structure of gyrase-DNA complex. To facilitate structural determination, we created a N-terminal ATPase domain-truncated GyrBA DBCC fusion protein that contains the C-terminal region of GyrB (residues 395-804) and the full-length GyrA (residues 1-875). The fusion protein has been successfully expressed and purified to homogeneity. Crystallization trials of this fusion protein in complexes with a 40-bp DNA duplex and antibacterial fluoroquinlones have been initiated.