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  • 學位論文

參與減數分裂之拓樸異構酶VI類似蛋白Spo11的功能與結構解析

Structural Study of Spo11: a Topoisomerase VI-like Protein Essential for Meiotic Recombination

指導教授 : 詹迺立
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摘要


有性生殖物種必須透過減數分裂來產生單套染色體的配子體細胞,並且在透過精卵結合回復到雙倍體狀態。在此過程中,子代傳承了父母雙方的遺傳物質,同時增加了遺傳多樣性來面對大自然中的諸多天擇壓力,讓生物個體更有機會存活並繁衍後代。減數分裂包含了一次的染色體複製和兩次的細胞分裂,依序達成同源染色體以及姊妹染色體的分離。在同源染色體分離的過程中,染色體會進行聯會與互換,稱為同源重組作用。同源重組不但能進一步增加遺傳多樣性,也同時讓染色體能順利向細胞兩極移動來進行分裂,確保配子體細胞擁有正確的染色體數目。此過程的缺失將會導致個體失去生育能力,或產生異常倍體細胞,使子代帶有基因缺陷,例如唐氏症 (Down syndrome)是由患者帶有三條21號染色體所導致。同源重組中雙股DNA互換必須由關鍵蛋白Sporulation-specific protein 11 (Spo11)催化產生計畫性DNA雙股斷裂 (programmed DNA double-strand breaks, DSBs)來啟動。Spo11的序列和出現在古生菌中的拓樸異構酶VI (Topoisomerase VI, Top6)的A次單元 (Top6A)具有很高的同源性,因此推測兩者間可能存在結構和催化機制的相似性。Top6由兩個A次單元以及兩個B次單元(Top6B)所組成具有活性的異質四聚體結構,因此推測Spo11會和也參與在同源重組中的Top6B同源蛋白,稱為Topoisomerase VI B-like (Top6BL)進行交互作用,形成類似的異質四聚體來執行催化作用。Spo11和Top6A會利用催化核心上高度保留的酪氨酸 (tyrosine)對DNA骨架進行親核性攻擊,產生DSB。但與Top6相比,由Spo11所催化的DSB是不可逆的,而是會透過同源重組來進行修復。除了催化核心Spo11以外,DSB的形成還需要其他多種蛋白的參與。在酵母菌Saccharomyces cerevisiae 中,Ski8會和Spo11形成複合體,並將之帶至細胞核之中,同時Ski8也是一個關鍵的鷹架蛋白,很有可能扮演著連接其他DSB相關蛋白的重要角色。此外,Top6BL對於DSB的催化也是不可或缺的,並也在演化上高度保留。然而這些DSB的相關蛋白在催化過程中的功能尚未被了解透徹,Spo11在分子層次上何和這些DSB相關蛋白進行交互作用以達成DNA的切割,都仍待釐清。 實驗室已經可以透過大腸桿菌蛋白表現系統,經過液相層析純化後,得到嗜熱真菌Myceliophthora thermophile (MYCTH)的Spo11-Ski8異質二聚體。然而經過多方嘗試後,依然無法得到MYCTH Spo11-Ski8的蛋白質晶體,又該複合體雖然具有結合DNA的活性,但卻缺乏切割活性,因此我們嘗試獲得MYCTH Top6BL蛋白來和Spo11-Ski8進行交互作用,以重建出具有切割活性的催化核心,並且提供另一種生成晶體的可能性。但透過膠體過濾液相層析的測試,並未觀察到Spo11-Ski8和Top6BL之間的交互作用,意味著我們取得的Top6BL可能錯誤折疊,或需要更多條件來形成具有切割活性的複合體。此外,我們也嘗試透過冷凍電子顯微鏡 (cryogenic electron microscopy, cryo-EM)解析MYCTH Spo11-Ski8之立體結構解析。然而初步的影像顯示,Spo11-Ski8在進行冷凍樣品製備時,複合體會有分離的現象。因此我們便透過glutaraldehyde讓蛋白間產生交叉連結 (cross-link),來穩定複合體結構,藉此解決複合體分離的問題。在中研院生化所的協助下,經過交叉連結的Spo11-Ski8經過影像分類和重構後,得到了解析度大約10 Å 的電子密度圖形,只能看到非常粗略的輪廓,還不足以解析蛋白質分子內部的結構。後續可以透過調整緩衝液的組成或是改善樣品均值性,來得到解析度更高的電子密度圖,以便後續的結構解析。

並列摘要


Meiosis is an essential process for the life cycle of sexually reproducing organisms by which diploid progenitor cells divide to produce haploid gametes. During meiosis prophase I, a specialized process termed meiotic recombination, which involves the establishment of physical connections between homologous chromosomes, is engaged to allow not only the formation of synaptonemal complex and chiasma to ensure accurate chromosome segregation but also the reshuffling of genetic materials to increase genetic diversity. Mechanistically, meiotic recombination is initiated by the formation of programmed double-strand breaks (DSBs) catalyzed by the sporulation-specific protein 11 (Spo11), an evolutionarily conserved enzyme that is homologous to the A subunit of topoisomerase VI (Top6). Due to the significant homology between Spo11 and Top6A, it has been proposed that these two proteins may share similar structural features and catalytic mechanism. Previous studies have revealed that Top6 functions as a heterotetramer composed of two A (Top6A) and two B (Top6B) subunits. The winged-helix DNA-binding (WHD) domain of Top6A harbors the catalytic tyrosine for executing DNA cleavage (DSBs) via a transesterification reaction. The TOPRIM domain of Top6A also plays an essential role in DNA cleavage by coordinating Mg2+ to facilitate transesterification reaction. Beside Spo11, at least another nine proteins are required for DSB formation in saccharomyces cerevisiae. Among them, the identified component termed topoisomerase VI-B-like (Top6BL) is particularly notable, due to the similarity with Top6 B subunit by comprising GHKL ATPase domain and transducer domain. The discovery of Top6BL suggested that, like Top6, Spo11 may also function in the context of an A2B2 heterotetramer. To date, the DNA cleavage activity of Spo11 has not been observed biochemically, and no structural information of Spo11 is currently available. To elucidate how Spo11 mediates DSBs during meiosis, the main goal of my thesis study is to determine the three-dimensional structure of Spo11 and reconstitute its DNA cleavage activity in vitro. Full-length Spo11 of Myceliophthora thermophila (MYCTH) was co-expressed with its direct binding partner Ski8 to obtain the Spo11-Ski8 heterodimer in soluble form. The Spo11-Ski8 complex exhibits DNA binding activity and appears suitable for further structural and biochemical studies. However, despite various attempts to crystallize the Spo11-Ski8 complex, including the construction of GST-tagged Spo11-Ski8 and protein surface entropy reduction by deletion or mutation of specific residues, the complex has eluded crystallization thus far. We also tried to determine the structure of Spo11-Ski8 by cryogenic electron microscopy (Cryo-EM). The preliminary data showed that Spo11-Ski8 complex was disrupted during the freezing process. To overcome this problem, we attempted to stabilize the complex by GraFix, a protein crosslinking method in which protein sample is exposed to glutaraldehyde gradient. The images of the sample after GraFix showed improved homogeneity and was used for 3D reconstruction to obtain a low-resolution EM map (about 10 Å). On the other hand, given the essential role of Top6BL in DSB formation, we attempted to reconstitute the ternary complex by pooling the purified Spo11-Ski8 and Top6BL together, but no interaction was observed between the two samples, as judged by the result of size exclusion chromatography. Several tasks are in progress toward a better understanding of key proteins involved in the initiation of meiotic recombination.

並列關鍵字

Spo11 Ski8 Top6BL Meiosis meiotic recombination DSB cryo-EM

參考文獻


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