臺灣博碩士論文加值系統

真核生物演化出特別的策略以濃縮DNA的體積，利用一段長度為147個鹼基對的DNA片段纏繞組蛋白形成基本且具重複性的結構，將其結構稱為核小體 (Nucleosome)。核小體依其結構的特性調控生物體內需DNA為模板的生合成反應，因此，核小體在基因表現上扮演著格外重要的角色。同時真核生物發展許多方式以改變核小體結構，而Chromatin structure remodeling complex (RSC, Remodel the Structure of Chromatin)具有調節核小體結構的活性。先前研究指出RSC能水解ATP獲得能量以擾亂核小體的結構，移動核小體在DNA上的位置，或是造成組蛋白八聚體脫離DNA的束縛。然而關於RSC如何調節核小體重組的機制和過渡態尚未解析清楚，本實驗利用單分子栓球技術 (TPM, Tethered Particle Motion)觀察RSC與核小體反應的過程，並著重於RSC導致組蛋白八聚體解離的機制進行探討。TPM實驗所得到的結果顯示: RSC利用ATP水解所得能量調節核小體結構並伴隨DNA環的產生，而ATP濃度上升影響了反應起始時間的提早、DNA環發生的頻率增加及DNA環之間的間隔時間隨之縮短。同時ATP存在的條件下能夠觀測組蛋白八聚體解離的現象，ATP濃度上升也導致解離的比例上升及解離時間的提前。然而，Nap1存在下會導致RSC調節組蛋白八聚體解離的比例下降。本實驗使用TPM實驗探討RSC調節核小體重組的機制，包括核小體移動，組蛋白八聚體解離，以及與其他因子之間的交互作用。

Eukaryotes evolved a specific strategy for DNA packaging. A repeating unit, nucleosome, consists of 147 base pairs of DNA segment wrapped histone core proteins. Nucleosome plays an important role in the regulation of DNA-dependent biosynthesis and gene expression by its structure. Chromatin structure remodeling complex (RSC, Remodel the Structure of Chromatin) is essential for the regulation of nucleosome structure in vivo. Previous researches indicated that RSC complex can hydrolyze ATP to perturb nucleosome structures resulting in nucleosome sliding and histone octamer ejection. However, the mechanisms and the transition state of RSC-mediated nucleosome remodeling are still unclear. Here we utilize tether particle motion (TPM) experiments to investigate the RSC-mediated nucleosome remodeling process in detail. RSC has been known to play an important role in DNA-dependent biosynthesis pathways. Therefore, we study the interplay between RSC and other factors, acceptor DNA and nucleosome assembly protein (Nap1). By using TPM experiments, the histone octamer ejection was observed during RSC-mediated nucleosome remodeling. Moreover, the high percentage of the RSC-mediated histone octamer ejection was observed under saturated ATP and in the presence of acceptor DNA. In contrast, the ratio of RSC-mediated histone octamer ejection decreased in the presence of Nap1. In this project, the whole reaction scheme of RSC-mediated nucleosome remodeling process, including nucleosome sliding and histone octamer ejection, can be depicted based on TPM results.

致謝 i
目錄 ii
表圖目錄 iv
中文摘要 1
英文摘要 2
第一章 緒論 3
1. 1 核小體 3
1. 1. 1 核小體結構及功能 3
1. 1. 2 核小體的動態 4
1. 2 RSC 5
1. 2. 1 重組蛋白家族 5
1. 2. 2 RSC結構與功能 7
1. 2. 3 RSC調節核小體重組的動態 8
1. 3 組蛋白伴隨蛋白Nap1 10
1. 3. 1 Nap1結構及功能 10
1. 3. 2 在生合成反應中與RSC的互動 11
1.4 單分子栓球實驗 11
1. 5 研究動機 12
第二章 實驗設計與材料製備 13
2. 1 蛋白質製備 13
2. 1. 1 RSC表現及純化 13
2. 1. 2 Nap1表現及純化 16
2. 2 DNA製備 18
2. 3 核小體製備 20
2. 4 玻片製備 22
2. 4. 1 Flow chamber製備 22
2. 4. 2 玻片表面修飾及DNA的固定 23
2. 4. 3 anti-digoxigenin修飾於220 nm標示小球之製備 24
2. 5顯微鏡架設及影像擷取 26
第三章實驗結果與討論 27
3. 1 活性測試 27
3. 1. 1 RSC 27
3. 1. 2 Nap1 28
3. 2 RSC調節核小體重組的動態 30
3. 2. 1 實驗設計 30
3. 2. 3 布朗運動值在時間內變化的事件定義 31
3. 2. 4 不同ATP濃度下RSC調節核小體重組的動態之活性比例及參數訊息 36
3. 3 接受者DNA參與RSC調節核小體重組的動態 41
3. 3. 1 實驗設計 41
3. 3. 2接受者DNA的參與影響核小體重組的動態 43
3. 4 Nap1參與RSC調節核小體重組的動態 45
3. 4. 1 實驗設計 45
3. 4. 2 核小體重組事件的變化 47
第四章總結與未來展望 51
4. 1 實驗總結 51
4. 2 未來展望 55
附錄 56
附錄 DNA601序列 61
附錄 利用Normal slide架設TPM實驗 62
Reference 65
圖1. 1 核小體的組成及結構。 4
圖1. 2 真核生物調節核小體動態的機制。 5
圖1. 3 重組蛋白蛋白家族及其區域分布。 6
圖1. 4 RSC與DNA和核小體結合之關係。 8
圖1. 5 重組蛋白調節核小體移動的模型。 9
圖1. 6 Nap1的二級與三級結構。 10
圖1. 7 Nap1存在下RSC調節核小體動態的變化。 11
圖1. 8 DNA長度對應布朗運動值之檢量線。 12
圖2. 1 TAP purification之流程圖。 15
圖2. 2 RSC純化膠圖。 15
圖2. 3 yNap1純化膠圖。 17
圖2. 4 PCR反應條件。 19
圖2. 5 PCR之洋菜膠片圖。 19
圖2. 6 773 bp 單核小體於分段下的分布之EMSA圖。 21
圖2. 7 Flow chamber之示意圖。 23
圖2. 8 觀測核小體動態之TPM實驗的架設。 24
圖2. 9 NHS/EDC crosslink reaction之機制。 25
圖3. 1 RSC調節202 bp單核小體動態之EMSA圖。 27
圖3. 2 yNap1調節核小體組裝之EMSA圖。 29
圖3. 3 以TPM實驗架設觀察RSC調節773 bp單核小體重組動態之示意圖。 30
圖3. 4 773 bp bare DNA與單核小體之間布朗運動值的差異。 31
圖3. 5 No event的時間軌跡圖。 32
圖3. 6 Loop event的時間軌跡圖及波峰之定義。 34
圖3. 7 Ejection event的時間軌跡圖。 35
圖3. 8 ATP濃度為0 mM的條件下，不同類型DNA的代表布朗運動時間軌跡圖及類型比例。 37
圖3. 9不同ATP濃度條件下代表布朗運動時間軌跡圖及類型比例。 38
圖3. 10不同ATP濃度下，Waiting time及Ejection time之指數遞減圖。 39
圖3. 11 不同ATP濃度下，Loop frequency、Loop-loop duration及Loop size之指數遞減圖。 41
圖3. 12以接受者DNA作為實驗對象，加入RSC調節核小體重組反應。 42
圖3. 13以TPM實驗架設觀察RSC調節773 bp單核小體重組動態之示意圖。 43
圖3. 14接受者DNA加入RSC調節核小體重組之代表布朗運動時間軌跡圖及類型比例。 44
圖3. 15接受者DNA加入RSC調節核小體重組反應之參數。 45
圖3. 16 Nap1參與轉錄的機制。 46
圖3. 17以TPM實驗架設觀察RSC調節773 bp單核小體重組動態之示意圖。 46
圖3. 18 yNap1加入RSC調節核小體重組之代表布朗運動時間軌跡圖及類型比例。 47
圖3. 19 yNap1加入RSC調節核小體重組反應之參數。 48
圖4. 1 RSC調節單核小體重組之機制圖。 53
圖4. 2 Nap1存在下RSC調節單核小體重組之機制圖。 54
圖S1 觀測核小體動態之TPM實驗的架設。 63

表2. 1 模板與引子 18
表 2. 2 PCR反應之反應條件 18
表2. 3 氯化鈉濃度梯度透析之條件 20
表3. 1 各個反應條件的類型比例 49
表3. 2 各個反應條件的參數訊息 50
表S1 藥品清單 56
表S2 緩衝溶液條件 57

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