梨形鞭毛蟲拓樸異構酶IIIα以及拓樸異構酶II結合蛋白1之分析與功能鑑定

Abstract

梨形鞭毛蟲是一種真核單細胞的原蟲類人畜共通寄生蟲，感染區域遍布全球。在生活史上分為滋養體以及囊體兩種型態：囊體透過水源或受汙染的食物進入人體，在小腸上壁形成較活躍的滋養體型態，大量分裂產生疾病，最後再次形成囊體排出體外。其主要症狀為輕微腹瀉、腹痛，脫水和營養不良等。無論是細胞複製，或是從染色體套數16N的囊體轉變為4N的滋養體的過程，遺傳物質DNA的複製、重組、轉錄等步驟都是極為重要且需要精準表現的，而DNA從原本穩定的超螺旋結構解開時，會因為緊密纏繞而遇到許多拓樸學上的問題，這時便需要拓樸異構酶的協助，才能使DNA表現正常的功能。此篇研究分為兩個部分： 第一部分為梨形鞭毛蟲拓樸異構酶IIIα (後面皆簡稱為gTOP3α)之研究。拓樸異構酶IIIα具有第一型拓樸異構酶功能，能夠切割單股DNA緩解DNA纏繞。在大部分真核生物體內，則會與RecQ family、RMI蛋白質形成RTR complex，協助增強DNA同源染色體交換以及DNA的修復作用。實驗室在先前發現了梨形鞭毛蟲的gTOP3α基因。先前已發現在大量表現gTOP3α後會降低囊體化時期的重要蛋白質cyst wall protein(cwp)表現量，且在gTOP3α的突變株中也會減緩對囊體壁蛋白質的抑制作用。此研究中，經過體外測試發現，gTOP3α蛋白質能夠與DNA進行結合，但在將gTOP3α C端的zinc finger結構刪除後，造成對DNA的結合能力大幅下降。我們發現在大量表現gTOP3α的梨形鞭毛蟲滋養體內，作為DNA損傷標記物Phospho-Histone H2A.X (ser139) (γH2AX)表現量上升，因此我們懷疑gTOP3α的大量表現和造成DNA損傷有關。於是我們在梨形鞭毛蟲WB蟲株的培養液中，加入導致DNA損傷的methyl methanesulfonate (MMS)藥物，發現在加入MMS的實驗組細胞產生明顯的死亡。透過realtime PCR發現，梨形鞭毛蟲體內和細胞ER stress的bip和mlf 的RNA及蛋白質表現量均有上升的現象；在參與減數分裂之同源重組(homologous recombination, HR)有關的梨形鞭毛蟲dmc1b基因的RNA也有顯著上升。由於真核生物在進行雙股DNA斷裂修復作用時，進行同源重組會需要topoisomerase IIIα的參與，而我們也發現gTOP3α及gTOP3β的RNA在加入MMS後表現出些微上升；而與DNA修復相關的TOPBP1也表現出上升，而協助斷裂DNA接合的Spo11 RNA也表現出些微上升。將大量表現gTOP3α的細胞株中加入MMS後，以免疫沉澱法(Immunoprecipitation, IP)也發現gTOP3α會與轉錄因子Myb2和轉錄因子WRKY進行直接或間接的結合。 我們的研究結果顯示，梨形鞭毛蟲gTOP3α參與在DNA修復作用，並能與調控囊體化相關轉錄因子產生交互作用。 第二部分為梨形鞭毛蟲拓樸異構酶II結合蛋白1 (gTOPBP1)之研究。首先我們從梨形鞭毛蟲基因體資料庫中找到了一標記為DNA修復基因，編號為6918。經過物種親緣關係和胺基酸結構預測後，發現這個基因與其他物種的拓樸異構酶II結合蛋白1 (Topoisomerase II-binding protein1, TOPBP1)的序列相似度較高，因此我們稱之為gTOPBP1。為了了解梨形鞭毛蟲 gTOPBP1是否具有我們已知的TOPBP1功能，像是活化DNA受損反應的重要上游蛋白質進行DNA修復、聚集並協助拓樸異構酶II活化等功能，以及在演化較早期的梨形鞭毛蟲體內，扮演什麼樣的角色。首先我們使用RT-PCR來偵測gTOPBP1 RNA在滋養體及囊體化時期的RNA表現量，發現gTOPBP1 RNA表現量在囊體化時期有增加。於是建構了gTOPBP1重組蛋白質的質體，轉染至梨形鞭毛蟲體內，以免疫螢光染色的方式觀察，發現在囊體化時期gTOPBP1會大量表現於細胞核內。我們也設計了將BRCT結構刪除的gTOPBP1m1，發現在滋養體及囊體化時期，gTOPBP1m1皆表現於細胞質內。接著利用西方墨點法分析在大量表現gTOPBP1和gTOPBP1m1的梨形鞭毛蟲滋養體及囊體化時期，對囊體壁蛋白質cwp1的影響，發現在滋養體時期gTOPBP1會抑制cwp1蛋白質的表現量，gTOPBP1m1也會抑制cwp1，但抑制效果較gTOPBP1低；在囊體時期，gTOPBP1會增加cwp1蛋白質的表現量，gTOPBP1m1也會增加cwp1表現，但效果較gTOPBP1低；在形成囊體的數量也得到與cwp1蛋白質改變相同的結果。而在電泳位移實驗中，也發現gTOPBP1能夠結合被EcoRI切割過的直線狀DNA，而gTOPBP1m1的結合能力則較gTOPBP1弱。 我們的研究結果顯示，梨形鞭毛蟲的gTOPBP1會在囊體化大量表現，能活化囊體化相關基因的表現，並且具有DNA結合的能力。

Giardia lamblia is a kind of eukaryotic protozoan zoonosis parasite that belongs to Excavata and spreads all over the world. Through the life cycle of Giardia, there are two forms, trophozoite and cyst. When cyst form transmits to host by contaminated water or food, it turns into the more active trophozoite form in duodenum. Trophozoite divides and causes symptom on hosts. Finally, the life cycle ends by turning into cyst again and pass to the environment. Disease that caused by Giardia called giardiasis. General symptoms are mild diarrhea, stomach, dehydration and malnutrition. During the replication of cell or transformation between cysts and trophozoites, there should be very precise control in the process of DNA replication, recombination and transcription. There will be some topology problems when DNA is relaxed from supercoiled form. Therefore, topoisomerase is needed to make a regular function for DNA. The thesis includes two parts: The first part is the research of Giardia Topoisomerase IIIα (gTOP3α). Topoisomerase IIIα has the type I topoisomerase functions, because it is able to cut single strand DNA to relax DNA. In most of eukaryotic creatures, topoisomerase IIIα will interact with RecQ family and RMI protein forming RTR complex to induce DNA homologous recombination and DNA damage repair. We have identified the gTOP3α gene of G. lamblia. According to previous study, overexpression of gTOP3α will reduce cyst wall protein (cwp), an important protein of encystation in Giardia. In this study, with Electrophoretic Mobility Shift Assay (EMSA), we found that gTOP3α protein has DNA binding function in vitro. The ability of DNA binding reduced when the C-terminal zinc finger domain was deleted. Furthermore, the amount of DNA damage marker Phospho-Histone H2A.X ser139 (γH2AX) increased by overexpression of gTOP3α. We wondered if there is a connection between gTOP3α expression and DNA damage. So we treated methyl methanesulfonate (MMS) that induced DNA damage in Giardia WB isolate and found MMS caused cell death obviously. Then we used realtime PCR to detect the expression changes, we found the product of bip and mlf genes which increase after ER stress also increased in both RNA and protein level. Dmc1b that plays a role in homologous recombination also increased in RNA level. Topoisomerase IIIα is required in eukaryotes when DNA double strand break is repaired by homologous recombination, we found gTOP3α and gTOP3β RNA increased slightly. The DNA repair gene TOPBP1, also increased in RNA level after treatment of MMS to Giardia. Spo11 which has functions in repairing of DNA breaks also increased in RNA level after treatment of MMS to Giardia. We suggest that gTOP3α has function in DNA repairing and interacts with encystation transcription factors. The second part is research of Giardia Topoisomerase II-binding protein 1 (gTOPBP1). We found a gene number 6918 is labeled as DNA repair gene by Giardia database. And we found this gene has similarity sequence with Topoisomerase II-binding protein1 (TOPBP1), therefore we named it as gTOPBP1. In most of eukaryotic creatures, TOPBP1 helps activation of ATM-ATR pathway to help DNA repair and interacts with Topoisomerase II. In order to know the role of gTOPBP1 in Giardia lamblia. First, we used RT-PCR to determine the RNA expression of gTOPBP1 in trophozoite and encystation stages. We found gTOPBP1 RNA was expressed more in encystation. Immunofluorescence assay revealed that gTOPBP1 was major localized in nucleus in encystation. We also designed a mutant with a deletion of BRCT domain, gTOPBP1m1. Immunofluorescence assay revealed that gTOPBP1m1 was major localized in cytosol in trophozoite and encystation stage. In the result of western blot, we found overexpression of gTOPBP1 reduced the expression of cwp1 in trophozoite stage. gTOPBP1m1 reduced less cwp1 than gTOPBP1 in trophozoite stage. Overexpression of gTOPBP1 induced the expression of cwp1 in encystation stage. gTOPBP1m1 induced less cwp1 than gTOPBP1 in encystation stage. The cyst formation test reveals the same results as cwp1 protein changing. By electrophoretic mobility shift assay, we found that gTOPBP1 have ability to bind linear form DNA cut with EcoRI. gTOPBP1m1 has less ability to bind DNA. To sum up, this study suggests that gTOPBP1 plays an important role in DNA damage repair and in induction of the cwp1 protein and encystation relating transcription factors.