嗜熱毀絲黴之拓樸異構酶VI類B次單元的結構與功能分析

Abstract

遺傳係指生物體繁衍後代時傳遞基因與性狀的現象，多數行有性生殖的物種會經由減數分裂以形成配子，再由雌雄配子的結合的而傳承生命，而在減數分裂的過程中會發生基因重組、使配子的遺傳多樣性更加豐富。真核生物中有一個高度保留、在起始減數分裂過程中不可或缺的蛋白，其名為Spo11；藉由催化轉酯反應，Spo11能在雙股DNA上形成斷裂，進而驅動基因的重組。在此過程中、Spo11會以其活性中心的酪胺酸攻擊DNA 5′端的磷酸形成磷酸二酯鍵，以共價鍵結的方式與DNA連結，同時釋放3′端的羥基，完成對DNA的切割作用；而以酪胺酸攻擊DNA 5′端的磷酸形成共價鍵的催化機制，與第二型拓樸異構酶VI切割DNA的催化機制相同，經由序列比對得知，Spo11與拓樸異構酶VI其中名為A次單元的蛋白具同源性。 拓樸異構酶VI廣泛存在於古生菌中，是一個由兩個A次單元及兩個B次單元所構成的異質四聚體酵素，負責催化DNA拓樸構型的改變，A次單元能與DNA結合並擁有切割能力，負責催化DNA的斷裂；B次單元含有負責水解三磷酸腺苷的GHKL功能域，以及傳導功能域，GHKL功能域水解三磷酸腺苷後會發生構型改變，此時連接A與B兩個次單元之間的傳導功能域可將B次單元的構型改變導引至A次單元而發生切割，使得拓樸異構酶VI具有完整的活性。鑒於拓樸異構酶VI須以A2B2的結構存在才有作用能力，推論Spo11這個類A次單元蛋白也可能是以A2B2四聚體的形式執行DNA切割。近期的研究指出，在體外實驗中，線蟲 (C. elegans)體內純化出的單體 (monomeric) Spo11，即便與DNA結合也不具切割單股或雙股DNA的能力。鑒於拓樸異構酶VI B次單元的同源蛋白，也被證實的確在老鼠 (Murine)與阿拉伯芥 (Arabidopsis)中存在，且為減數分裂過程中不可或缺的重要蛋白，以生化功能的分析及結構的相似度推測此類B次單元的蛋白極有可能與Spo11交互作用。 我們利用上述類B次單元的序列進行比對，發現嗜熱毀絲黴中兩個可能為類B次單元的基因，其一之C端區域較為完整，序列也較長。此段基因可合成一段含有499個胺基酸的多肽鏈，我們在其N端引入可移除之GST標籤以方便純化。藉由先前的實驗得知，Spo11只有在Ski8鷹架蛋白(scaffold protein)的存在下才能以可溶蛋白的形式出現，然而Spo11-Ski8複合體雖然具有與DNA結合的能力，卻無法切割DNA，此現象是否可藉由拓樸異構酶VI 類B次單元與Spo11形成複合體而有所回復呢？因此本研究的主要目的之一就是獲得具有酵素能力的拓樸異構酶VI 類B次單元並且做結構及功能性分析。我首先使用共價偶聯glutathione穀胱甘肽管柱層析 (batch column)純化GST融合之類B次單元，然而在沖提之後，發現此蛋白似乎會與樹脂緊密結合上而無法取得，更改沖提條件依然無法獲得蛋白。在推測會Spo11會與類B蛋白交互作用的前提下，嘗試利用黏附在樹脂上之融合蛋白獲得Spo11-Ski8複合體，而相對的，亦可利用純化之Spo11-Ski8複合體的方式獲得拓樸異構酶VI 類B次單元，然而至今尚無任何交互作用測試可證實嗜熱毀絲黴之拓樸異構酶VI 類B次單元與Spo11-Ski8複合體之間的交互作用。 我們仍期望樸異構酶VI 類B次單元能與Spo11或Spo11-Ski8複合體形成複合體，雖然目前對於身為鷹架蛋白的Ski8尚有許多不了解，然而樸異構酶VI 類B次單元在體內實驗被證實是與Spo11直接作用的蛋白，並且一旦此蛋白功能缺失，細胞內即不會產生雙股斷裂。因此，本實驗所表現出的拓樸異構酶VI 類B次單元在構型上正確與否尚有討論空間。拓樸異構酶VI 類B次單元必須正確摺疊方能執行其生物功能，摺疊不正確的蛋白在純化過程中，有極高的機率會暴露出其疏水性片段而與樹脂之間產生疏水性交互作用而無法沖提下來，樹脂上殘留無法沖提之拓樸異構酶VI 類B次單元顯示可能為蛋白本身構型未正確摺疊所致，因而影響後續與Spo11-Ski8複合體之間的交互作用的實驗結果，未來將釐清此疑義方能回歸此研究的中心主旨: Spo11究竟是如何切割DNA的？

Heredity is the term used to describe the maintenance of parental traits and genes during reproduction. Most species that undergo sexual reproduction would go through meiosis to form haploid gamets, subsequent combination of male and female gamets leads to the formation of a new life. Meiotic recombination is a process through which genes from homologous chromosomes are intentionally recombined to increase genetic diversity of the gamets. This crucial recombination process is initiated by the controlled introduction of DNA double-strand breaks (DSBs). Spo11, a conserved eukaryotic protein, has been recognized as a triggering factor that catalyzes DSB formation. Mechanistically, Spo11 performs a transesterification reaction between its active site tyrosine and the DNA 5′-phosphate and becomes covalently linked to the DNA 5′-end with comcomittant release of a hydroxyl group on the 3′-end . Such a mechanism of DNA cleavage is the same as the one employed by topoisomerase VI (TopoVI), a type IIB DNA topoisomerase. Indeed, sequence analysis revealed that Spo11 is homologous to the A-subunit of topoisomerase VI (TopoVI). TopoVI is a tetrameric DNA-manipulating enzyme composed of two A (TopoVI-A) and two B (TopoVI-B) subunits found mainly in archaea and plants. The TopoVI-A subunit is responsible for DNA binding and cleavage. The TopoVI-B subunit possesses a GHKL domain capable of binding and hydrolysis of ATP and a transducer domain that bridges the cross-talks between the GHKL domain and the TopoVI-A subunit. Given that TopoVI functions as an A2B2 tetramer, it has been speculated that Spo11 would either dimerize or form a TopoVI-like heterotetramer to exhibit its DNA cleavage activity. Recently, a protein homologous to the TopoVI-B subunit has been identified in Murine and Arabidopsis. Biochemical analysis and structural modeling revealed that this TopoVI-B-like protein (TopoVI-BL) is structurally similar to TopoVI-B and may interact with Spo11. Sequence alignment shows the existence of two genes that may be homologous to TopoVI-BL in Myceliophthora thermophila (MYCTH). The longer one has a more complete C-terminal domain and encodes a polypeptide of 499 amino acids. We introduced a GST-tag to the N-terminus of this longer TopoVI-BL and obtained a fusion protein that may be purified more readily. Previous experiments have shown that Spo11 is only soluble when co-expressed with a scaffolding protein Ski8. While the purified recombinant MYCTH Spo11-Ski8 complex can bind to DNA, no cleavage activity was observed. We speculated that TopoVI-BL may interact with Spo11-Ski8 complex to activate the cleavage ability to Spo11. Thus, a major aim of my thesis research is to obtain active TopoVI-BL for structural characterization and functional analysis. After attempting to purify by GST-fused TopoVI-BL using glutathione-charged resin, however, TopoVI-BL remained tightly associated with the beads and cannot be eluted from the resin. Different elution conditions made little effect. A subsequent strategy we employed for testing the interaction between TopoVI-BL and the Spo11-Ski8 complex was using the beads coated with either TopoVI-BL or Spo11-Ski8 complex to pull down the other. Unfortunately, currently none of the results showed the presence of an interaction between them. Still, TopoVI-BL and are expected to form a complex with Spo11 and/or Spo11-Ski8 complex. Although little is known about Ski8 other than its role as a scaffold protein, TopoVI-BL is identified in vivo as a direct binding partner of Spo11 and, without TopoVI-BL, double-strand breaks would not occur during meiosis. Thus, there may be some issues regarding whether the purified TopoVI-BL is correctly folded. A misfolded protein has a greater tendency to expose hydrophobic residues and form non-specific hydrophobic interaction with the resin. The failure to elute TopoVI-BL from the beads indicate that TopoVI-BL may not fold appropriately. And the non-native structure further affects its interaction with Spo11-Ski8 complex. Therefore, it remains hopeful that a complex formed by TopoVI-BL and Spo11-Ski8 can be obtained to address to understand how Spo11 cleaves DNA.