DNA拓樸異構酶可解決DNA在進行轉錄、複製、重組、修復等生理作用時產生的一些拓樸結構問題。舉例來說，DNA在轉錄及複製時形成的超螺旋結構會影響聚合酶的前進，導致轉錄及複製無法順利進行；DNA在複製時發生的雙股連鎖則會造成染色體的不穩定，因此DNA拓樸異構酶在維持DNA的正常構造及基因體的完整性上扮演著很重要的角色。拓樸異構酶可利用其酵素活性中心的酪胺酸對DNA的磷酸雙酯鍵進行親核性攻擊，藉由催化可逆的轉酯作用，造成DNA短暫的斷裂，使另一單股或雙股的DNA得以通過缺口，達成DNA構型的改變，接著第二次的轉酯作用便可以使DNA缺口重新連接。拓樸異構酶依結構和機制的不同主要可分為Type I和II，Type I可造成單股DNA斷裂而Type II則會引發雙股DNA斷裂，此二型酵素又可再進一步細分為IA和IB、以及IIA和IIB兩種亞型。拓樸異構酶VI是Type IIB的唯一成員，主要存在於古生菌和部分植物。拓樸異構酶VI是由兩個A次單元和兩個B次單元組成的異質四聚體，A次單元負責結合、切割與黏合DNA，而B次單元負責結合、水解ATP，並可輔助DNA的捕捉與搬運。雖然IIA和IIB兩種拓樸異構酶亞型之間具有演化上的關聯性，且催化機制亦非常類似，然而拓樸異構酶VI的A次單元與Type IIA酵素之功能對應區域無論在胺基酸序列和立體結構上皆有著顯著的不同，且兩者切割DNA後形成之產物也不同，因此我們認為IIA和IIB兩種亞型結合、切割DNA的機制勢必有所不同。此外，本研究的另一個有趣的背景就是拓樸異構酶VI的A次單元及B次單元的同源蛋白皆被報導參與了減數分裂時的同源重組，其作用機制則仍待釐清。鑒於目前為止仍沒有任何關於拓樸異構酶VI與DNA結合的結構資訊，因此本研究希望能以X射線結晶學解析拓樸異構酶VI與DNA的複合體結構。先前本實驗室已成功建立大量表達以及取得高純度且具完整活性之嗜熱菌拓樸異構酶VI蛋白的操作流程，但尚無法培養出蛋白晶體，因此我嘗試加入第二型拓樸異構酶的抑制劑doxorubicin與etoposide以增加蛋白與DNA形成之複合體的穩定度，但結果顯示兩種藥物對蛋白的解旋活性並沒有顯著影響，因此這個策略的可行性偏低。之後我也試著將拓樸異構酶VI可能影響晶體形成且較不保留的序列做去除，但發現如此一來蛋白的解旋活性下降了一些，再經由凝膠電泳遷移分析(EMSA)的實驗判斷解旋活性下降可能是因為突變蛋白與DNA結合變弱所致。除此之外，我也試著單獨表現並純化拓樸異構酶VI的A次單元及B次單元，並且以EMSA分析二者與DNA的交互作用，發現兩者皆具有良好的DNA結合能力，因此我也進行養晶條件的測試，希望取得A次單元及B次單元與DNA形成之的複合體結構。

Topoisomerases can resolve DNA topological problems that arise during fundamental biological processes such as replication, transcription, and recombination. For example, a progressing replisome will introduce positive DNA supercoils and intertwining daughter chromosomesahead of and behind the replication fork, respectively. These higher order DNA structures may cause stalling of replication forks and promote the formation of DNA breaks, which results in chromosome instability. Therefore, topoisomerases play an essential role in maintaining the genome integrity and stability. Topoisomerases alter DNA topology by catalyzing the passage of a single- or double-strand DNA segment through a transiently formed single- or double-strand break (DSB) in another. These topoisomerase-mediated DNA breaks are characterised by a phosphotyrosyl bond that links the broken DNA strand to the enzyme. Topoisomerases are classified into two types: type I produces single-strand DNA break while type II cleaves both DNA strands. Type II enzymes are further divided into two subfamilies, IIA and IIB: Type IIA topoisomerases are present throughout bacteria and eukaryotes, whereas the type IIB topoisomerase, topoisomerase VI (Topo VI), mainly exists in archaea, plants, green and red algae, and in a few bacteria. Topo VI functions as a heterotetramer composed of two copies of each VI-A and VI-B subunit. VI-A is responsible for DNA binding and cleavage and VI-B is involved in ATP binding and hydrolysis.
Despite similarity in function, there are crucial mechanistic differences between type IIA and IIB topoisomerase. It is especially noteworthy that the DNA binding regions of the two topoisomerase families are structurally distinct. The structural differences may reflect significant variations in their interaction with DNA. Moreover, SPO11 and MTopoVIBL, the evolutionarily conserved eukaryotic orthologs of VI-A and VI-B, respectively, is known to trigger meiotic recombination by producing chromosomal DSBs. A better understanding on the molecular mechanisms controlling this process may be achieved through structural analysis of Topo VI.
To date, there is no structural information available to explain the interaction between Topo VI and DNA. Therefore, the long-term goal of this study is to determine the structure of Topo VI-DNA complex by X-ray crystallography. To this end, we reconstituted and purified the Topo VI holoenzyme from a thermophilic archaea, Nanoarchaeum equitans, for structural and functional analyses. We found that Topo VI exhibitd an ATP- and catalytic tyrosine-dependent relaxation activity. We have also tested whether the topoisomerase II inhibitors doxorubicin and etoposide can inhibit the relaxation activity of Topo VI. It appears that these two drugs exhibit no effect on Topo VI. We next investigated the interaction between Topo VI and short substrate DNA duplex or long linearized pRYG using EMSA. Our EMSA data demonstrate that the recombinant Topo VI effectively interacts with both types of DNA. We also initiated crystallization screens on the Topo VI-DNA binary complex. To facilitate crystallization, a truncated Topo VI was subsequently produced by removing the less conserved surface loop regions. However, the mutant Topo VI exhibited reduced relaxation activity and DNA binding affinity compared to wild-type enzyme. To understand better the functional properties of Topo VI-A and Topo VI-B, we indivisually purified these two Topo VI components for this study. Interestingly, our EMSA analyses demonstrate that not only Topo VI-A but also Topo VI-B binds our substrate DNA. Crystallization trials for the Topo VI-A and Topo VI-B are currently underway.

謝誌 I
摘要 II
Abstract IV
縮寫表 VI
目錄 VII
圖目錄 X
表目錄 XI
一、前言 1
1.1.　DNA拓樸結構問題 1
1.2.　拓樸異構酶之分類與功能 2
1.3.　拓樸異構酶VI之結構與催化機制 4
1.4.　第二型拓樸異構酶之比較 5
1.5.　拓樸異構酶VI之同源蛋白對於減數分裂的重要性 6
1.6.　研究動機 7
二、材料與方法 8
2.1.　蛋白質表現系統 8
2.1.1. 表現質體建構 (Construction of expression plasmids) 8
(1) pETDuet-NEQ542-144 8
(2) pET-21b-NEQ542 8
(3) pET-21b-NEQ144 9
(4) pETDuet-NEQ542-shortened NEQ144 (Topo VI-J) 9
(5) pETDuet-NEQ542 (Y93F)-NEQ144 9
(6) pRYG supercoiled DNA 10
2.1.2. 轉型作用 (Transfomation) 10
2.1.3. 蛋白表現量測試 10
2.1.4. 蛋白之大量表現 11
(1) pETDuet-NEQ542-144 (Topo VI) 11
(2) pET-21b-NEQ542 (Topo VI-A) 12
(3) pET-21b-NEQ144 (Topo VI-B) 12
(4) pET-21b-NEQ542-shortened NEQ144 (Topo VI-J) 12
(5) pETDuet-NEQ542(Y93F)-NEQ144 (Topo VI-Y93F) 12
2.2.　蛋白質純化 13
2.2.1. 破菌與蛋白萃取 13
2.2.2. 液相層析 (liquid chromatography) 13
(1) pETDuet-NEQ542-144 (Topo VI) 蛋白純化 13
(2) pET-21b-NEQ542 (Topo VI-A) 蛋白純化 15
(3) pET-21b-NEQ144 (Topo VI-B) 蛋白純化 15
(4) pETDuet-NEQ542-shortened NEQ144 (Topo VI-J) 蛋白純化 16
(5) pETDuet-NEQ542(Y93F)-144 (Topo VI-Y93F) 蛋白純化 16
2.3.　蛋白質分析及定量 17
2.3.1. 蛋白膠體電泳分析 17
(1) SDS-PAGE 膠體製備 17
(2) 蛋白電泳分析 17
(3) 染色及退染 (destain) 18
2.3.2. 蛋白質濃縮定量 18
2.4.　蛋白質活性測試 18
2.4.1. 電泳遷移率改變實驗 (electrophoresis mobility shift assay，EMSA) 18
2.4.2. relaxation assay 19
2.5.　蛋白質晶體培養 19
2.5.1. 養晶樣品製備 20
2.5.2. 養晶前處理 20
2.5.3. 預結晶試驗 (pre-crystallization test) 21
2.5.4. 養晶方法 21
2.5.5. 養晶條件 21
2.6.　共養晶之DNA 22
2.6.1. 雙股DNA形成 (duplex formation) 22
三、結果 23
3.1.　蛋白質表現測試 23
3.1.1. Topo VI-A表現系統 23
3.1.2. Topo VI-B表現系統 23
3.1.3. Topo VI-J表現系統 24
3.2.　蛋白質純化 24
3.2.1. Topo VI純化 24
(1) 鎳離子親和性層析法 (Nickel column) 25
(2) 膠體過濾層析 (GFC) 25
3.2.2. Topo VI-A純化 25
(1) 鎳離子親和性層析法 (Nickel column) 25
(2) 膠體過濾層析 (GFC) 26
3.2.3. Topo VI-B純化 26
(1) 鎳離子親和性層析法 (Nickel column) 26
(2) 離子交換層析 (ion exchange chromatography) 27
(3) 膠體過濾層析 (GFC) 27
3.2.4. Topo VI-J純化 27
(1) 鎳離子親和性層析法 (Nickel column) 28
(2) 膠體過濾層析 (GFC) 28
3.2.5. Topo VI (Y93F)純化 29
(1) 鎳離子親和性層析法 (Nickel column) 29
(2) 膠體過濾層析 (GFC) 29
3.3.　蛋白質活性測試 30
3.3.1. Topo VI蛋白解旋活性測試 30
(1) Topo VI與IIA酵素抑制藥物之作用 30
(2) Topo VI與其突變型蛋白活性比較 31
3.3.2. 受質DNA之設計 31
3.3.3. 電泳遷移率改變實驗 (EMSA) 32
(1) Topo VI 32
(2) Topo VI-A 32
(3) Topo VI-B 33
(4) Topo VI-J 34
(5) Topo VI (Y93F) 34
3.4.　蛋白質晶體培養 35
3.4.1. Topo VI-DNA複合體之養晶測試 35
3.4.2. Topo VI-A-DNA複合體之養晶測試 35
四、討論 36
4.1.　蛋白質表現 36
4.2.　蛋白質純化 37
4.3.　蛋白質活性分析 38
4.4.　蛋白質晶體培養 40
圖 41
表 67
參考文獻 77

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