人類DNA拓樸異構酶III的純化與生化特性分析

Abstract

拓樸異構酶普遍存在於所有細胞，負責解決各種因DNA代謝過程，例如：複製、轉錄、染色體分離、……等，所造成DNA結構的拓樸學問題。近年來發現人類細胞有兩種拓樸酶III，分別被稱為hTOP3α和hTOP3β，愈來愈多的證據說明，這兩種同功酶可能在細胞中執行不同的功能，為了進一步探討同功酶的特異性，我們首先使用桿狀病毒與細菌表現系統來大量表達hTOP3α和hTOP3β，然後進行蛋白質純化。純化出的酵素在試管中測試其生化特性，結果發現採用不同受質DNA明顯影響了重組hTOP3α和hTOP3β的活性表現：兩者對一般帶負超螺旋的質體DNA僅具微弱解螺旋能力；但兩種同功酶都能順利將超負超螺旋受質解開成負超螺旋。上述觀察與前人對果蠅Top3的實驗結論相符。簡言之，本篇論文成功大量表達了hTOP3α和hTOP3β同功酶，並且建立了一個快速、容易且有效的純化方式，純化出的酵素將可被應用於未來更多的研究。 此外，之前已有報導證明在大腸桿菌中，過量表達Tnp將會抑制TopA；於酵母菌的研究亦顯示，過量表達Tnp也會抑制ALT端粒延長機制。綜合以上兩項實驗結果，以及TopA與hTOP3結構上的高度相似性，還有TOP3在ALT路徑扮演之角色，我們提出Tnp會抑制hTOP3的假設。為證實此假設，我們建構帶有Tnp、Inh、55aa的質體，並將之送入酵母菌或哺乳動物細胞以利進一步的研究。

Topoisomerases are ubiquitous enzymes found in almost all cells and play important roles in many biological processes, such as DNA replication, transcription, and chromosome condensation by regulating the topology of DNA. Two isoforms of topoisomerase III, hTOP3alpha and hTOP3beta, have been recently identified in human cells. Accumulating evidences indicated that hTOP3alpha and hTOP3beta might exhibit distinct cellular functions. In order to further clarify isozyme-specific activities, we first employed both baculoviral and bacterial expression systems to produce large amounts of enzymes for purification. The biochemical properties of purified enzymes were then tested in vitro. using negative supercoiled or hyper-negative supercoiled substrates. Despite of a rather weak relaxation activity against usual plasmid DNA, purifie hTOP3alpha and hTOP3beta showed their preferences for hyper-negative supercoiled structure which might have more single-stranded regions exposed. This result is in consistent with experiments done with Drosophila Top3. Taken together, we successfully overexpressed both hTOP3 isozymes and also developed a fast, easy, and efficient purification protocol. More studies could be done with these purified products in the future. It has been proved that overexpression of Tnp in E. coli would result in titration of TopA. The previous research done in S. cerevisiae also led to the conclusion that Tnp could inhibit ALT pathway. Because of the structure similarity between TopA and hTOP3, and the role Top3 played in ALT pathway, the hypothesis that Tnp might work through inhibiting Top3 was arised. We then constructed plasmids encoding Tnp, 55aa, and Inh. The recombinant plasmid will be sent into yeast or mammalian cells for further examination.