探討人類酪胺酸DNA磷脂酶在拓樸異構酶可切性複合體之處理路徑上所扮演的角色

Roles of Human Tyrosyl-DNA Phosphodiesterases in Processing Topoisomerase Cleavable Complexes

黃詩勻(Shih-Yun Huang)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03116

摘要

拓樸異構酶(topoisomerase I/II/III, TOP I/II/III)可以藉催化DNA短暫的斷裂和重新黏合而解決DNA的拓樸結構問題。另外，拓樸酶亦被證明為治療癌症及抗生素的藥物標靶；此類藥物可以阻止酵素催化的DNA重新黏合，進而穩定拓樸異構酶可切性複合體(topoisomerase cleavable complex, TOPcc)，造成DNA斷裂及細胞死亡。TOPcc是一個DNA和拓樸酶以共價鍵連結的複合體(蛋白質上的酪胺酸和DNA上的磷酸共價連結)；值得注意的是，DNA斷點是被包覆在蛋白質裡的，因此必須經過細胞處理路徑(cellular processing pathway)讓斷點裸露出來以進行修補及其他訊息傳遞。TOP1會經由酪胺酸和DNA 3’端的磷酸形成共價鍵造成單股DNA斷裂；TOP2則是經由酪胺酸和DNA 5’端的磷酸形成共價鍵造成雙股DNA斷裂。此外，許多報導指出，人類酪胺酸DNA磷脂酶(human Tyrosyl-DNA Phosphodiesterase 1/2, hTDP1/2)可以藉由切斷拓樸酶和DNA間的共價鍵而使DNA斷點裸露出來，才能啟動DNA損傷反應及路徑(DNA damage response, DDR)。然而，我們對於人類細胞hTDP1/2處理TOP2cc的功能及特異性以及hTDP1/2和細胞處理路徑(TIP : 轉錄起始的處理，RIP : DNA複製起始的處理)間的關係依然不清楚。為了釐清這些問題，我們利用RNA干擾技術、強制蛋白表現以及藥物處理調控…等方法來探討hTDP1/2對於TOPcc所引發的DNA damage所造成的影響以及DNA的傷害反應。我們的結果證明，在人類細胞中hTDP1參與在TOP1cc的修復過程，而hTDP1和hTDP2皆參與在TOP2cc的修復過程。此外，在修復TOP2cc所造成的DNA損害的過程當中，hTDP1和hTDP2可能參與在TIP和RIP的路徑中，且作用在TOP2cc上的酵素降解之後，切掉最後的磷酸酪胺酸共價鍵。此外，缺少hTDP1或hTDP2的細胞對於針對TOP2的藥物皆有較高的感受性。綜合以上實驗結果，我們的研究證實hTDP1/2在解決TOP2cc所造成的DNA斷裂中所扮演的角色；此外，TOP和TDP藥物的合併使用或許也可以更有效治療癌症。

關鍵字

拓樸異構酶拓樸異構酶可切性複合體 ； 人類酪胺酸DNA磷脂酶細胞處理路徑 ； DNA損傷反應 ； 轉錄起始的處理 ； DNA複製起始的處理

並列摘要

Topoisomerases (TOP1/2/3) can solve the topological problems of DNA through transient breakage and religation reactions and has become excellent targets of anticancer drugs. Drugs targeting topoisomerases induce the formation of topoisomerase cleavable complex (TOPcc) in a special form of protein-embedded, phosphotyrosyl-linked DNA breakage and hence blocking the accessibility of signaling and repair proteins. During the formation of TOPcc, TOP1 and TOP2 covalently links to DNA though a 3’-phosphotyrosyl bond and 5’-phosphotyrosyl bond, respectively. Tyrosyl-DNA phosphodiesterases (TDP1/2) have recently been reported to process the hidden DNA breakage in TOPcc into uncovered DNA lesions by mediating the cleavage of enzyme-DNA phosphotyrosyl linkage. However, the specificity to 3’- and 5’-phosphotyrosyl linkage and corresponding functions of hTDP1/2 in processing of TOP1/2cc remain ill defined. Toward this aim, we used RNA interference (RNAi) technology and overexpression system to investigate the roles of hTDP1/2 in the TOP1/2cc-mediated DNA breakage and DNA damage responses (DDR). In addition, the potential relationship among hTDP1/2, transcription- and replication-initiated processing (TIP and RIP) pathways are also studied. We found that hTDP1 contributes to the repair of TOP1cc while hTDP1 and hTDP2 both participate in the repair of TOP2cc in human cells. Moreover, our results also suggest that hTDP1/2 are involved in TOP2cc-mediated DDR that are activated by transcription- and replication-initiated processing pathway. Consistently, both hTDP1- and hTDP2-deficient cells are hypersensitive to TOP2-targeting drugs. Together, our studies not only demonstrated the distinctive roles of hTDP1/2 in the processing of TOP1/2cc but also suggest a novel combination therapeutic strategy of using TOP1/2- and TDP1/2-targeting agents.

並列關鍵字

topoisomerase ； topoisomerase cleavable complex ； DNA Phosphodiesterase ； cellular processing pathway ； DNA damage response ； transcription- and replication-initiated processing pathways

參考文獻

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