拓樸異構酶 VI 類似蛋白Spo11 之功能與結構解析

Towards Structural and Functional Analysis of Spo11, a Topoisomerase VI-like Protein

李易庭(Yi-Ting Lee)

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(2016年)

https://doi.org/10.6342/NTU201603103

摘要

自然界中行有性生殖之個體，係藉由減數分裂使親代與子代間的染色體數目保持恆定，以確保物種基因體的穩定性。同時，在減數分裂的過程亦發生了同源染色體片段的互換與重組，增加配子乃至群體之遺傳多樣性，此為生物產生變異以適應環境變化的重要機制。
生物體中利用Spo11，此「類第II型拓樸異構酶蛋白」(type II DNA topoisomerase-like protein)催化染色體之同源重組作用的發生。根據序列比對，Spo11與Topoisomerase VI的A次單元具有同源性，且透過相似機制進行DNA之剪切。於第一次減數分裂前期，Spo11會進入細胞核並以序列中高度保留的酪胺酸 (tyrosine)針對DNA進行親核性攻擊，形成磷酸酪胺基連接 (phosphotyrosyl linkage)並引發不可逆之DNA雙股斷裂 (double-strand break, DSB)後，起始重組酶的運作而使重組作用得以順利進行。雖然身為起始同源重組作用的關鍵因子，Spo11並無法單獨執行DNA的剪切，尚需多個蛋白參與作用。以Saccharomyces cerevisiae為例，Ski8蛋白即被視為Spo11的直接作用夥伴，二者能形成穩定的複合體並自細胞質遷移至細胞核中，穩定了Spo11和染色體間的結合。Ski8亦具有鷹架蛋白的特性，可扮演橋接的角色，調控蛋白複合體的聚合並招募其餘成員進入細胞核作用，如Rec102與Rec104等。然而由於缺乏結構資訊，致使目前對於Spo11之催化DSB形成的機制僅有初步認識，亦尚未釐清各類蛋白如何與Spo11交互作用，共同參與DSB的形成。
在嘗試表現各物種之Spo11蛋白而無顯著進展後，本研究目前以嗜熱真菌Myceliophthora thermophile (MYCTH)之Spo11作為主要實驗對象，有別於一般物種，嗜熱真菌的蛋白具有相對穩定的特性，利於純化實驗以及結構分析。另外基於S. cerevisiae的研究結果，我也嘗試將MYCTH Spo11與Ski8進行共表達，以獲取穩定且大量之MYCTH Spo11。本研究利用大腸桿菌作為蛋白表達系統，經液相層析與分子篩管柱純化後，可得水溶性之MYCTH Spo11-Ski8異質二聚體，再由活性試驗確認蛋白在30˚C環境下具有最佳結合DNA的能力，且在鎂離子存在時可造成環形質體DNA的拓樸構形改變；我亦利用多種養晶試劑篩選此異質二聚體之晶體生成條件，截至目前，尚未觀察到晶體之生成。後續實驗將嘗試共表現其餘蛋白複合體之成員，盼能有效重組MYCTH Spo11之切割DNA的活性並增進培養出晶體之可能性。

關鍵字

減數分裂 ； 同源重組作用 ； DNA雙股斷裂 ； Spo11 ； Ski8

並列摘要

Meiosis is an essential process for sexual reproduction and contributes to the long-term survival and fitness of eukaryotes. During meiosis, chromosomes undergo one round of amplification followed by two consecutive cell division events, leading to the transformation of one diploid cell into four haploid gametes, such that the number of chromosomes can be restored upon fertilization. Proper chromosome segregation at the first meiotic division is crucial for preventing fertility problems, birth defects and cancer and it requires recombination-mediated formation of linkages between homologous chromosomes. This so-called meiotic recombination is essential for increasing genetic diversity during sexual reproduction and is initiated by programmed double-strand break (DSB) at the beginning of meiotic prophase I. Sporulation-specific protein 11 (Spo11), a meiosis-specific type II DNA topoisomerase-like enzyme that is evolutionarily conserved from yeast to mammals, is known for its central role in meiotic recombination. Sequence analysis revealed that Spo11 is homologous to the A subunit (the DNA-binding and cleavage domain) of archaeon topoisomerase VI (Topo VI) but exhibits no obvious homology with the B subunit (the ATPase clamp domain). Therefore, it is possible that Spo11 may mediate DNA cleavage to initiate meiotic recombination, whereas the hallmark DNA strand passage and religation activities of Topo VI are not required. Base on the catalytic mechanism of type II topoisomerases, Spo11 most likely cleaves DNA with the formation of a covalent 5’-phosphotyrosyl linkage between the 5’ DNA end using the catalytic tyrosine residue resides in the winged-helix domain of each monomer, and by functioning as a dimer, Spo11 can simultaneously break both strands to produce DSBs. Previous studies have shown that Spo11 can indeed covalently attach itself to short oligonucleotides with different lengths, resulted from asymmetric cleavage by endonucleases. Although Spo11 is the catalytic center for the DSB formation in meiosis, it does not work alone. In budding yeast, at least nine other accessory proteins are required for the completion of this process. However, the structures of Spo11, either alone or in complexes with other binding partners have not been determined, since it has proved difficult to purify functional Spo11 protein. To better understand the biochemical activity of Spo11, we attempt to perform structural analysis on the full-length Spo11 of Myceliophthora thermophile (MYCTH), which can be successfully expressed in soluble form in Escherichia coli (E. coli). Unfortunately, the DNA binding and cleavage assays demonstrated that the recombinant Spo11 protein, when expressed alone, likely existed in an inactive state. In contrast, when co-expressed with its direct binding partner Ski8, large amount of soluble MYCTH Spo11-Ski8 heterodimer can be prepared. EMSA assay further demonstrated that DNA duplexes of various lengths can be retarded by the heterodimer, with optimal activity being observed at 30˚C, whereas purified MYCTH Ski8 lacks such an activity. This finding indicates that MYCTH Spo11 can be produced in E. coli in its active form in the presence of MYCTH Ski8. Moreover, relaxation of negatively supercoiled plasmid DNA was observed when incubated with the Spo11-Ski8 heterodimer in the presence of Mg2+, which may be explained by a Spo11-mediated DNA cleavage event. Crystallization trials for the MYCTH Spo11-Ski8 heterodimer is currently underway.

並列關鍵字

meiosis ； meiotic recombination ； DNA double-strand break ； Spo11 ； Ski8

參考文獻

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