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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹迺立 | zh_TW |
| dc.contributor.advisor | Nei-Li Chan | en |
| dc.contributor.author | 顏予晨 | zh_TW |
| dc.contributor.author | Yu-Chen Yen | en |
| dc.date.accessioned | 2021-07-11T15:37:12Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-09 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79020 | - |
| dc.description.abstract | 自然界中行有性生殖之生物，需透過減數分裂來產生僅具單套染色體的配子細胞，並經由受精作用將來自父母雙方的遺傳物質結合並傳承，此關鍵過程對於增加子代之遺傳多樣性至關重要。除了親代染色體的合併，減數分裂時，同源染色體之間還會發生聯會與互換，稱為重組作用；同源染色體間的交聯使其能以朝向細胞兩極的位向進行分離，而確保配子細胞中具有正確的染色體數目，此作用的缺失將導致子代遺傳物質的異常，嚴重者甚至可使胚胎無法順利發育為個體。Sporulation-specific protein 11 (Spo11) 能催化DNA雙股斷裂 (double-strand break, DSB) ，在減數分裂中為啟動DNA重組作用的關鍵蛋白，其序列與拓樸異構酶VI (topoisomerase VI, TopoVI) 的A次單元 (TopoVI-A) 高度相似。TopoVI由兩個A次單元及兩個B次單元 (TopoVI-B) 所構成，因此，推測Spo11極可能會形成同質二聚體，並利用其高度保留的酪胺酸 (tyrosine) 針對DNA骨架行親核性攻擊，形成磷酸酪胺基鍵結 (phosphotyrosyl bond) ，因而產生不可逆的DSB，使後續同源重組作用得以進行。雖然Spo11是催化DSB形成的核心蛋白，但除了本身以外，尚需要多種蛋白共同參與方能展現其完整的DSB催化活性；在Saccharomyces cerevisiae之中，Ski8能夠與Spo11形成穩定複合體，接著從細胞質遷移到細胞核中，幫助Spo11結合至染色體；Ski8亦可能作為鷹架蛋白，橋接DSB相關的蛋白複合體以調控Spo11對DNA的切割活性。近年來的研究，更發現了過去未知的TopoVI-B相似蛋白家族，稱為TopoVI-B-Like (TopoVI-BL) 蛋白，與Spo11行交互作用之餘，更能協助其進行二聚化，組成如同TopoVI的異質四聚體結構而催化DSB。然而，基於Spo11蛋白純化製備上的困難，對於Spo11執行DSB的分子機制尚不明朗，其如何與TopoVI-BL及Ski8行交互作用也仍然未知。
本研究使用之蛋白序列來自Myceliophthora thermophile (MYCTH)，此物種為生長於高溫環境的真菌，其蛋白具有相對較高之熱穩定性，利於表現與純化。目前利用大腸桿菌之蛋白表達系統，經過液相層析純化後，已能夠獲得穩定而具有良好水溶性的MYCTH Spo11-Ski8異質二聚體。電泳遷移速率試驗顯示，MYCTH Spo11-Ski8能夠穩定結合DNA，卻未見其擁有切割DNA、或是改變DNA之拓樸構形的能力；因此，我們亦嘗試MYCTH TopoVI-BL的表達與純化測試，期盼在MYCTH TopoVI-BL與Spo11-Ski8蛋白共同存在之下，能使MYCTH Spo11展現DSB的催化活性。我們亦使用多種晶體培養試劑，針對MYCTH Spo11-Ski8進行結晶試驗，但目前仍未見到蛋白晶體形成；為了增加晶體培養成功率，我檢視不同緩衝劑、鹽類及其它添加劑對蛋白水溶性的影響，並測試能使MYCTH Spo11-Ski8異質二聚體相對穩定的酸鹼值，進而使緩衝液成分最佳化。此外，我以MYCTH Spo11為基礎，建構Glutathione S-transferase (GST) 融合蛋白，並成功獲得GST-Spo11與Ski8之蛋白複合體，盼能夠進一步改善蛋白水溶性以及晶體培養的成功率；意外的是GST-Spo11/Ski8複合體在活性測試中顯示其可能具有DNA切割之能力，後續將透過實驗進一步地驗證此結果。 | zh_TW |
| dc.description.abstract | Meiosis is an essential process in all sexually reproducing organisms, which allows the expansion of genetic diversity within a population. During meiosis, DNA replication is followed by two rounds of cell division to produce four haploid gametes. Accurate segregation of chromosomes must be ensured to prevent random disjunction and aneuploidy, which may lead to embryonic death, birth defects and cancer. In particular, meiotic recombination between homologous chromosomes, including the formation of synaptonemal complex and chiasmata, enables them to be oriented properly on the spindle and thus segregated correctly, and this process is initiated by a programmed formation of double-strand breaks (DSBs) at the beginning of meiosis I prophase. Sporulation-specific protein 11 (Spo11), an evolutionarily conserved enzyme, is known for its critical role in the formation of DSBs. Sequence alignment revealed that Spo11 is homologous to the A subunit (the DNA-binding and cleavage domain) of archaeal topoisomerase VI (TopoVI), a type IIB topoisomerase composed of two A (TopoVI-A) and two B (TopoVI-B) subunits. Based on the similarity between Spo11 and TopoVI-A, it was proposed that at the onset of meiotic recombination, Spo11 would dimerize to execute DNA cleavage through the formation of a phosphotyrosyl linkage between the catalytic tyrosine residue, located in the winged-helix domain of each monomer, and a phosphate group of the DNA backbone, thus producing DSBs. However, neither the dimerization nor the DNA cleavage activity of Spo11 has been observed biochemically. In this regard, the newly discovered protein termed TopoVI-B-Like (TopoVI-BL), which shares sequence and structural similarity with the TopoVI-B by harboring the GHKL ATPase and transducer domains, suggests the function of Spo11 may require a physical association with TopoVI-BL. Structural modeling predicts that TopoVI-BL and Spo11 may form a heterotetramer like TopoVI holoenzyme to promote DSB formation. Studies conducted in yeast further revealed that at least nine other proteins may cooperate with Spo11 to perform DNA cleavage. No structural information of Spo11, either alone or in complex with other accessory proteins, is currently available due to the difficulties in purifying functional Spo11 protein.
To clarify 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 E. coli upon co-expressing with its direct binding partner Ski8. The electrophoretic mobility shift assay (EMSA) revealed that purified MYCTH Spo11-Ski8 heterodimer exhibits unambiguous DNA binding ability. However, no cleavage activity was observed with either linear or supercoiled circular DNA as substrate. In light of the discovery of TopoVI-BL, we also attempted to obtain recombinant MYCTH TopoVI-BL protein in soluble form, which is expected to interact with Spo11-Ski8 and stimulate the DNA cleavage activity of Spo11. To obtain structural information of MYCTH Spo11-Ski8, crystallization trials of the heterodimer has been initiated. Furthermore, buffer screening and pH tolerance test were conducted to improve the solubility of MYCTH Spo11-Ski8, in hoping to enhance the probability of obtaining protein crystals. We also constructed GST-tagged Spo11 and successfully purified large amount of GST-Spo11 fusion protein in complex with Ski8, which will be used for crystallization screen. Unexpectedly, a Mg2+-dependent DNA cleavage activity was observed when GST-Spo11/Ski8 was incubated with linear or supercoiled DNA. This exciting new finding will be further validated through other experimental approaches. In addition, we have purified and crystallized the Ski8 monomer proteins in two different conditions. X-ray diffraction experiments are currently underway. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:37:12Z (GMT). No. of bitstreams: 1 ntu-107-R05442029-1.pdf: 5286439 bytes, checksum: f7ed1611ea29e39f9d28425b0ee0b995 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 謝誌 I
摘要 II Abstract IV 縮寫表 VII 目錄 VIII 圖目錄 XI 表目錄 XII 一、前言 1 1.1 減數分裂的生理意義與同源重組過程 1 1.2 Spo11執行DNA雙股斷裂的機制 2 1.3 與Spo11共同作用的蛋白複合體 3 1.4 DNA雙股斷裂發生的時機與位置 5 1.5 Topoisomerase VI之結構與催化機制探討 6 1.6 TopoVI-BL蛋白家族的全新發現 8 1.7 研究動機 9 二、 材料與方法 10 2.1 蛋白質表現系統 10 2.1.1 質體建構 10 2.1.2 表現蛋白菌種 11 2.1.3 轉形作用 (Transformation) 12 2.1.4 蛋白質小量表現測試 13 2.1.5 蛋白質大量表現與收集 17 2.2 蛋白質純化 19 2.2.1 破菌與蛋白萃取 19 2.2.2 液相層析法 (Liquid Chromatography) 19 2.3 蛋白質分析及定量 27 2.3.1 蛋白質膠體電泳分析 27 2.3.2 蛋白質鑑定 28 2.3.3 蛋白質濃縮定量 29 2.4 蛋白質活性測試 29 2.4.1 電泳遷移率變化分析 (Electrophoretic Mobility Shift Assay，EMSA) 29 2.5 蛋白質穩定性測試 31 2.5.1 pH值對蛋白複合體之影響 31 2.5.2 緩衝溶液成分篩選 (Buffer Screen) 31 2.6 蛋白質晶體培養 32 2.6.1 預結晶試驗 (Pre-crystallization Test, PCT) 32 2.6.2 蛋白質結晶方法與條件篩選 33 2.7 蛋白質晶體之X-ray繞射 34 2.7.1 MYCTH Ski8 (SeMet-labelled) 晶體冷凍保護 (Cryo-protection) 34 2.7.2 MYCTH Ski8 (SeMet-labelled) 晶體X-ray 繞射數據收集 34 三、實驗結果 35 3.1 蛋白質小量表現測試 35 3.1.1 GST-MYCTH Spo11表達系統 35 3.1.2 GST-MYCTH Spo11與Ski8共表達 (Co-expression) 系統 35 3.1.3 MYCTH TopoVI-BL表達系統 36 3.1.4 MYCTH TopoVI-BL與Spo11共表達系統 36 3.1.5 MYCTH TopoVI-BL與Ski8 & Spo11共表達系統 37 3.2 蛋白質純化 37 3.2.1 MYCTH Spo11與Ski8 (Spo11-Ski8) 37 3.2.2 GST-MYCTH Spo11與Ski8 (GST-Spo11/Ski8) 39 3.2.3 MYCTH Ski8 (SeMet-labelled) 41 3.3 蛋白質活性測試 42 3.3.1 MYCTH Spo11-Ski8之DNA結合與切割活性測試 43 3.3.2 GST-MYCTH Spo11-Ski8之DNA結合與切割活性測試 43 3.4 蛋白質穩定性測試 44 3.4.1 pH值變化對MYCTH Spo11-Ski8之影響 44 3.4.2 MYCTH Spo11-Ski8蛋白緩衝溶液成分篩選 45 3.4.3 GST-MYCTH Spo11-Ski8蛋白緩衝溶液成分篩選 46 3.5 蛋白質晶體培養 46 3.5.1 MYCTH Ski8 (SeMet-labelled) 46 3.6 蛋白質結構解析 47 四、討論 48 4.1 蛋白質表現與純化 48 4.2 蛋白質活性測試 49 4.3 蛋白質穩定性測試 50 4.3.1 MYCTH Spo11-Ski8 50 4.3.2 GST-MYCTH Spo11-Ski8 50 4.4 研究方向與展望 50 圖 52 表 85 參考文獻 91 附錄 96 | - |
| dc.language.iso | zh_TW | - |
| dc.title | 嗜熱毀絲黴之Spo11、Ski8及TopoVI-BL蛋白功能與結構解析 | zh_TW |
| dc.title | Towards Structural and Functional Analysis of Spo11, Ski8 and TopoVI-BL from Myceliophthora thermophila | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 曾秀如;徐駿森 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | 減數分裂,同源重組作用,DNA雙股斷裂,Spo11,Ski8,TopoVI-BL, | zh_TW |
| dc.subject.keyword | meiosis,meiotic recombination,DNA double-strand break,Spo11,Ski8,TopoVI-BL, | en |
| dc.relation.page | 100 | - |
| dc.identifier.doi | 10.6342/NTU201801835 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-15 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
| dc.date.embargo-lift | 2023-10-09 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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