人類拓樸異構酶Шα及Шβ之純化與功能解析

Toward Structural Studies of Human Topoisomerase Шα and Topoisomerase Шβ

謝詔名(Chao-Ming 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(2012年)

https://doi.org/10.6342/NTU.2012.01923

摘要

DNA雙股螺旋結構的骨架由五碳糖與磷酸雙酯鍵所組成，因此具有彈性並可以進行小幅度的構形改變，故在DNA進行生理反應時，會因結構中出現的張力而導致超螺旋的生成，進而抑制反應的進行，或造成基因的不穩定。此外，DNA在進行修復或重組時，也會出現糾纏的中間物。上述這些DNA的拓樸問題 (DNA topological problems )，需要拓樸異構酶的活性才能順利解決。
拓樸異構酶在改變DNA的拓樸狀態時，會利用活性中心的酪胺酸，與DNA的磷酸鍵結形成phosphotyrosyl bond，進而打斷DNA。第一型拓樸異構酶打斷單股DNA，第二型則打斷雙股DNA。人類的拓樸異構酶三 (hTOPШ)在分類上屬於第一型的拓樸異構酶，且有Шα與Шβ兩種亞型，顯示在演化過程中TOPШ出現了功能分歧，兩者可能在生理功能上扮演不同的角色。目前已知hTOPШα會與BLM (RecQ-helicase family)及RMI1形成複合體，並利用TOPШα切斷與接合DNA的活性來解開與DNA重組之中間物Holliday Junction相似的DNA構型，可參與在DNA雙股斷裂與複製叉停滯的修復、端粒延長 (ALT)，或是解開兩個複製叉會合時的DNA纏繞。因此，人類的TOPШα具有重要的生物功能。目前對TOPШβ的研究結果較為缺乏，只知道其與TOPШα差異在於不能與BLM以及RMI1形成複合體；在醫學上的研究則發現其活性可能與乳癌有關，但具體的機制卻仍不甚了解。
因此，本研究希望利用結構學的角度，來探討幾個問題：1. 人類TOPШα與TOPШβ功能上的差異為何？ 2. 為什麼TOPШα可以和BLM及RMI1形成複合體，但TOPШβ卻不行？ 3.與大腸桿菌的TOPШ序列比對後，發現人類TOPШ在C端多出了一段序列，其生理功能為何？ 4. 解出人類TOPШα及TOPШβ的結構後，或可設計TOPШα的抑制劑，來阻斷癌細胞利用ALT 途徑進行端粒延長。本篇論文將對 hTOPШα及hTOPШβ蛋白表現量及可溶性的問題，進行改善。

關鍵字

拓樸異構酶IIIα ； 拓樸異構酶IIIβ ； BLM ； RMI1 ； 端粒延長

並列摘要

Cellular DNA exists in double-helical form as the most stable structure. Due to the intrinsic flexibility of its phosphodiester backbone, however, the DNA can undergo conformation change. In the presence of strains, usually caused by over- or under-winding of the base pairs, supercoils will be introduced that may inhibit DNA transactions or induce genome instability. Moreover, the repair of damaged DNA usually leads to intertwined intermediates. To maintain the topological homeostasis and genome integrity, many types of topoisomerases have evolved to solve various topological problems.
Mechanistically, topoisomerases use the active site tyrosine to cleave the phosphodiester DNA backbone by forming the phosphotyrosyl bond. Depending on whether cleavage occurs at one or both DNA strands, topoisomerases can be divided into type I and II, respectively. Human topoisomerase III (TOPIII) is classified as a type I toposiomerase, and possess two TOPIII isoforms – IIIα and IIIβ, suggesting that they may have specialized functions.
Previous studies indicate that hTOPШα is a part of BLM core complex that also includes BLM (RecQ-helicase family) and RMI1 (RecQ-mediated genome instability protein 1). The DNA cleavage activity of hTOPIIIα is required for the resolution of Holliday Junction-like structures that occur during the repare of double strand break, stalled replication fork, alternative lengthing of chromosome pathway (ALT), and converging of replication forks. And the involvement of hTOPIIIα in ALT pathway suggests a potential anticancer therapeutic strategy. Unlike hTOPIIIα, hTOPIIIβ does not interact with BLM and RMI1. Though hTOPIIIβ is known to associate with M phase chromosomes, but its in vivo functions in human have remained poorly defined.
The long term goal of this study is to understand the cellular functions of hTOPIIIα and hTOPIIIβ, and there are several outstanding questions to be addressed. 1. What are the functional differences between the two isoforms? 2. How can hTOPIIIα interact with BLM and RMI1 but hTOPIIIβ cannot? 3. Sequence alignment of hTOPIIIα and the E. coli TOP3 revealed that hTOPIIIα has an additional C-terminal domain, whose functional requires further structural and biochemical studies. 4. The structures of hTOPIIIα and hTOPIIIβ would be helpful for developing inhibitors that can be used to block the ALT pathway. We have constructed expression plasmids for both proteins, and the suitable conditions for expressing these two recombinant proteins have been extensively tested in E. coli. We are currently resolving the protein solubility issues as well as resort to yeast for protein expression.

並列關鍵字

Topoisomerase IIIα ； Topoisomerase IIIβ ； BLM ； RMI1 ； ALT pathway

參考文獻

1. J. J. Champoux, DNA topoisomerases: structure, function, and mechanism. Annual review of biochemistry 70, 369 (2001).

2. J. C. Wang, Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Quarterly reviews of biophysics 31, 107 (1998).

3. H. Y. Wu, S. H. Shyy, J. C. Wang, L. F. Liu, Transcription generates positively and negatively supercoiled domains in the template. Cell 53, 433 (1988).

5. K. Kirkegaard, G. Pflugfelder, J. C. Wang, The cleavage of DNA by type-I DNA topoisomerases. Cold Spring Harbor symposia on quantitative biology 49, 411 (1984).

6. C. Sissi, M. Palumbo, Effects of magnesium and related divalent metal ions in topoisomerase structure and function. Nucleic acids research 37, 702 (2009).