拓樸異構酶VI與去氧核醣核酸複合體之結構解析

Toward structural analysis of Topoisomerase VI-DNA complex

謝子晴(Tzu-Ching Hsieh)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902874

摘要

拓樸異構酶可以解決DNA進行複製、轉錄、重組、修復等生理作用時所衍生的一些拓樸結構問題，細胞若缺乏拓樸異構酶的活性會導致DNA超螺旋或交聯等拓樸結構無法被解開，使得DNA或RNA聚合酶無法順利作用，造成轉錄或複製的停滯，最後使細胞發生凋亡，因此可知拓樸異構酶在維持細胞生理作用上扮演不可或缺的角色。拓樸異構酶的主要功能在於解開DNA超螺旋構型、DNA重組時雙股間的纏繞、染色體聚集以及解開纏繞或交聯的DNA。舉例來說，進行細胞分裂時，複製後之染色體必須要平均分配至兩個子細胞中，此時第二型拓樸異構酶可以藉由切割在一段雙股DNA中產生缺口，然後讓另一段雙股DNA通過此缺口來解開複製後交聯的姊妹染色體。
拓樸異構酶解決DNA拓樸構型的方式首先會與一條雙股DNA結合並誘導單股或雙股斷裂，接著藉由DNA旋轉或使另外一段單股或雙股DNA穿過斷裂處，最後再將原本產生斷裂的DNA黏合(religation)，藉此解開DNA拓樸構型。而依據DNA產生單股或雙股斷裂的差異，拓樸異構酶被分為第一型與第二型兩大類，其中各型中又因結構以及功能上的不同可再細分為A亞型和B亞型，本研究的主角拓樸異構酶VI (Top VI)就是屬於第二型拓樸異構酶的B亞型(type IIB topoisomerase)。相較於A亞型主要存在於細菌和真核生物中，B亞型則多分布在古生菌、植物、藻類以及一些細菌中。
拓樸異構酶VI是有兩個A次單元和兩個B次單元所組成的異質四聚體，其中A次單元負責執行DNA的結合與可逆性切割；B次單元主要是由ATPase和負責傳遞構型變化的transducer二個功能區所組成，可執行DNA的捕捉和傳送。而有關拓樸異構酶VI目前公認的催化機制是一段雙股DNA(G-segment DNA)會先和A次單元結合，之後A次單元上具有催化活性的Tyrosine會攻擊DNA骨架上5’端的磷酸基團形成磷酸二酯鍵 (phosphodiester bond)，藉此切割DNA，並產生5’-phosphotyrosine共價複合體；接著ATP會與B次單元結合，使兩個B次單元形成雙倍體(dimer)，此時Top VI整體會呈現閉合的狀態，藉此捕捉另外一段雙股DNA(T-segment DNA)，而當ATP水解後會帶動整體蛋白構型的轉變，使得已被酵素切割的G-segment DNA分開而產生開口，使T-segment DNA可以經由這個開口被傳送出去，最後開口會再被黏合，完成DNA拓樸構型的改變。但直到現在關於Top VI如何和G-segment DNA結合進而產生切割和再黏合，以及DNA gate產生的方式目前仍屬未知，所以本研究的目標就是希望透過解析Top VI-DNA複合體的立體結構，以了解Top VI和DNA產生交互作用以及切割的方法，並探討DNA gate產生的方式。
本研究所使用的Top VI來自於古生菌Nanoarchaeum equitans (NEQ)，此菌種具有嗜熱和嗜鹽的特性，先前的酵素活性分析已證實NEQ Top VI同樣也具有嗜熱以及嗜鹽的特性。嗜熱方面此酵素可以耐高溫至75℃，並且於此溫度下能展現很好的解旋活性，也發現若在純化過程中利用加熱的方式去除雜蛋白，可以得到很好的純化效果；嗜鹽方面已知NEQ Top VI在環境中NaCl濃度低於250 mM時容易產生沉澱，進一步發現它可於1 M NaCl高鹽環境下還能保有四聚體結構和功能。而根據先前優化好的方式進行純化、然而在測試過所有結晶條件後尚未發現Top VI長晶的條件。而先前亦曾針對Top VI表面較具動態的部位設計一些突變，希望可以增加結構穩定度幫助養晶，但在500 mM NaCl環境下純化後進行養晶卻有高比例的沉澱產生，推測是因為蛋白樣品和養晶試劑做等比例混和後因稀釋導致鹽濃度減半所致，不利於晶體產生。若提高蛋白樣品鹽濃度到750 mM以及1 M後確實能有效降低養晶時沉澱的形成，也得到若干Top VI可能長晶的條件，藉由X光繞射分析後也初步確認應該不是鹽類，因此開始嘗試二維微調改變緩衝液的酸鹼值以及沉澱劑的比例、加入additives、microseeding等方式，希望藉此改善晶體的品質。
鑒於本研究的主要目標為鑑定Top VI-DNA結合的複合體結構，然而實作時為了降低養晶時沉澱發生而增加鹽濃度，可能會因此干擾Top VI與DNA的結合，所以也測試Top VI在高鹽環境下與DNA結合的能力，確定在高鹽的養晶環境下DNA仍然會與蛋白結合，未來將透過繞射及結構分析確認養出的晶體是否為Top VI-DNA複合體。

關鍵字

拓樸異構酶VI ； 拓樸結構 ； 第二型拓樸異構酶

並列摘要

Topoisomerases can solve topological problems caused by cellular DNA transactions such as replication, transcription, recombination, chromosome condensation and segregation. For examples, newly replicated daughter chromosomes are interlinked and therefore must be separated to opposite ends of the dividing cell, such that each daughter cell receives one copy of the genome. During this processes, Topoisomerases IV can disentangle the replicated daughter strands by generating a transient double-stranded break (DSB) in one DNA duplex that allows another duplex to pass through.
Mechanistically, topoisomerases resolve topologically strained and entangled DNA structures by first introducing a single or double-stranded break in one DNA segment followed by transporting another DNA segment through the break. Depending on whether one or both DNA strands are cleaved during the catalytic cycle, these enzymes are classified into type I and type II, respectively. Moreover, each type can be further divided into A or B subtype based on structural and functional distinctions. Type II topoisomerases are divided into two subfamilies, IIA and IIB: type IIA topoisomerases are found in bacteria and eukaryotes, whereas the type IIB topoisomerases are presented in archaea, plants, algae and some bacteria. Topoisomerase VI (Top VI), the subject of my thesis research, belongs to type IIB topoisomerase.
Top VI exists as an A2B2 heterotetramer. Like conventional type II enzymes, Top VI catalyzes DNA topological changes by producing a DSB, passing another duplex DNA through the break, then resealing the cleaved DNA. The A subunit is responsible for DNA cleavage, whereas the B subunit is involved in ATP binding and hydrolysis. However, structural information regarding how Top VI interacts with DNA is yet unavailable, thus the mechanistic details of Top VI-mediated DNA cleavage and resealing of DNA has remained largely uncharacterized. The main goal of my thesis research is to determine the structures of Top VI–DNA complexes.
This work uses Top VI from the halophilic archaean Nanoarchaeum equitans (NEQ) as the subject. Earlier studies have revealed that the NEQ Top VI is prone to precipitate under medium-salt (< 250 mM) condition, which disfavors crystallization. To overcome this problem, I tried to purify NEQ TopoVI in the presence of higher salt concentrations, 750 mM and 1 M NaCl. Moreover, I found that NEQ Top VI can form functional tetramer in these conditions and exhibit robust relaxation and DNA binding activity, suggesting that the structure and activities of Top VI are intact in high salt concentrations. Preliminary crystals have been obtained using the high salt-stabilized protein samples in the presence of substrate DNA and AMPPNP. The crystallization conditions are currently being refined to improve crystal quality using various approaches such as additives and detergents screen, microseeding, heating purification or change protein concentration etc.

並列關鍵字

Topoisomerase VI ； DNA topology problem ； Type II topoisomerase

參考文獻

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3. Rybenkov, V.V., et al., Simplification of DNA Topology Below Equilibrium Values by Type II Topoisomerases. Science, 1997. 277(5326): p. 690-693.

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