人類第二型拓樸異構酶α亞型之結構解析

Abstract

拓樸異構酶是生物體內不可或缺的酵素之一，不論原核或真核生物，都需要透過拓樸異構酶改變DNA拓樸構形的活性，移除細胞複製、轉錄及染色體重組等作用中產生的超螺旋、扭結或連鎖等阻礙反應進行的DNA結構，使DNA代謝作用得以順利完成。 拓樸異構酶依照其於反應過程中造成DNA斷裂數目的差異可分為兩大型：第I型酵素催化DNA單股斷裂而改變1個單位的DNA連結數 (Lk)；第II型可使DNA雙股同時斷裂而讓Lk增減2個單位。每一型拓樸異構酶又依照序列相似性及催化機制再分為A、B兩個子類，而本篇論文研究的主題為屬於IIA型的人類拓樸異構酶IIα。目前對於IIA型拓樸異構酶的研究顯示，此類酵素的催化過程需要仰賴ATP，透過酵素活性中心的酪胺酸 (tyrosine) 對一段稱為G-segment的雙股DNA之磷酸雙酯鍵 (phosphodiester bond) 進行親核性攻擊形成磷酸酪胺基連接 (phosphotyrosyl linkage)，並將此DNA切開而產生缺口，再利用與ATP的結合與水解促進另一段稱為T-segment的雙股DNA穿過此缺口，此過程涉及酵素四級結構大幅度的變化。然而目前對於反應不同階段所發生的構形變化尚需更多的結構資訊，才能對酵素的作用機制有更深入的認識。本研究中我們進行了近乎全長序列的人類拓樸異構酶IIα與DNA複合體的結構解析：首先使用酵母菌表現此蛋白，並且經液相層析與分子篩管柱進行純化，最後以活性試驗確認蛋白可形成結構正確並且具有正常催化活性的二聚體。 目前我們已經獲得人類拓樸異構酶IIα與DNA複合體的蛋白晶體，並且進行晶體培養條件的微調後，經由X-ray繞射數據及運算軟體得到解析度 (resolution) 約6.6 Å的蛋白結構。將此結構與先前已發表的酵母菌拓樸異構酶II (PDB：4GFH) 進行疊合比較，可發現我們解出的人類拓樸異構酶IIα之ATPase功能域 (domain) 與主要催化G-segment DNA結合及切割的活性區域 (DNA binding and cleavage core，DBCC) 相距較遠，猜測兩者應該分別對應於催化機制中不同的結構狀態。此外，由於我們的晶體結構可同時容納G-segment與T-segment DNA的存在，因此可能提供酵素藉由構形改變催化T-segment通過G-segment過程的重要資訊，所以我們設計了一段具有35-mer並且5’與3’分別模擬為G-segment及T-segment的DNA，希望未來能與人類拓樸異構酶IIα共同結晶，進而以X-ray繞射解得酵素同時與G-segment和T-segment產生交互作用的結構。

Topoisomerases are essential enzymes ubiquitously present in eukaryotes, archaebacteria, and eubacteria. With their activities in manipulating DNA topology, these enzymes can resolve DNA entanglements and supercoils and are known to play critical roles in many cellular DNA transactions, including replication, transcription, recombination and chromatin remodeling. Topoisomerases can be classified into two types based on whether one or both DNA strands of a duplex are cut during a catalytic event: type I and type II topoisomerases introduce single or double strand breaks, respectively, to alter the DNA linking number in steps of either one and two. Each type of topoisomerase can be further divided into two sub-families (A or B) depending on sequence similarities and differences in catalytic mechanisms. Higher eukaryotes encode 6 functionally distinct topoisomerases: Top1, Top1mt, Top2α, Top2β, Top3α, and Top3β. Our studies focus on the human topoisomerase IIα (hTop2α), which belongs to the type IIA category. Previous studies show that type IIA topoisomerases possess an ATP-dependent DNA passage activity. The enzyme binds to and cleaves one DNA duplex (termed the G-segment) via the formation of phosphotyrosyl linkages between a pair of catalytic tyrosines and DNA backbone phosphodiester bonds, thus opening up a gate on the G-segment for transporting another DNA duplex (termed the T-segment) through the break. The binding and hydrolysis of ATP are central for Top2α function, which drives protein conformational changes to allow the capture and transport of T-segment. However, structural basis underlying the transitions between the various conformational states of Top2α has remained unclear. To better understand the whole mechanism, we attempt to perform structural analysis on a near full length hTop2α in complex with DNA. The recombinant protein can be successfully expressed in yeast strain BCY123 and purified to homogeneity. Size exclusion chromatography revealed that hTop2α produced by yeast exists as dimers. Functional analysis further demonstrated that the recombinant hTop2α retains wild-type-like catalytic activities and sensitivity toward topoisomerase-targeting drugs. We have recently obtained crystals of the hTop2α-DNA binary complex that diffract to about 6.6 Å resolution. The diffraction quality of these crystals was optimized by refining the crystallization conditions to facilitate structural determination. Preliminary structural analysis shows that the ATPase domain and the DBCC (DNA binding and cleavage core) domain of hTop2α are further apart compared to the structure of yeast Top2 (PDB: 4GFH). Given that the T- and G-segment can be accommodated simultaneously in our structure, it appears that hTop2α may be trapped in a new conformational state during the enzymes catalytic cycle. Therefore, we designed a 35-mer DNA substrate, which mimics the presence of both the G-segment and the T-segment for co-crystallization with hTop2α. We expect to elucidate how hTop2α interacts with G-segment and T-segment simultaneously by performing X-ray diffraction analysis of hTop2α in complex with the 35-mer DNA.