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姓名 王懷元(Huai-Yuan Wang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 引發體蛋白PriA和DnaD在DNA複製重啟之結構與功能分析
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摘要(中) 在DNA複製時，總是會遇到一些DNA受損或DNA結合蛋白質，他們可能會使複製體 (replisome) 離開複制叉DNA並形成廢棄的複制叉DNA。在bacillus subtilis中，它們演化出一種稱為“DNA複製重啟”的途徑來恢復 (restore) DNA複製。DNA複制的重啟是由PriA、DnaD和DnaB來完成。首先PriA會去辨識這個廢棄的複制叉DNA並招來DnaD和DnaB來組裝成一個引發體 (primosome) 。一旦引發體組裝好了，它就會將複製體裝載到DNA上並重新啟動DNA複製。然而，這些引發體蛋白質在DNA複製重啟中的分子機制仍舊不清楚。為更加了解其生化特性和引發體組裝活性，我們表達了從Geobacillus stearothermophilus和Streptococcus pneumoniae中克隆priA和dnaD基因並純化了該蛋白質。我們使用螢光極化 (fluorescence polarization) 來檢測蛋白質和蛋白質以及蛋白質和DNA的交互作用。結果表明，PriA對DnaD具有高親和力，兩種蛋白質均具有DNA結合能力。為了解釋複製重啟的機理模型並闡明它們的調節作用，我們試圖通過X射線晶體學研究結構和功能之間的關係，並通過多波長異常衍射解出2.5Å的DnaD的晶體結構。然而，我們沒有觀察到DnaD的 C端區域，可能是因為其撓動的特性。這些蛋白質結構和生化特性的初步結果使我們更進一步了解引發體組裝機制。
摘要(英) During the DNA replication elongation, this process is always encountering some problems that might eject the replication complex and form an abandoned replication fork DNA such as DNA damage. In bacillus subtilis, they evolve a pathway call “DNA replication restart” to resume DNA replication. DNA replication restart is done with several proteins PriA, DnaD and DnaB. First, PriA can recognize this abandoned replication fork DNA and recruit others primosomal proteins DnaD and DnaB to assemble a primosome. Once primosome is assembled, it loads a replisome onto DNA and restarts DNA replication. However, the molecular machinery of these primosomal proteins in DNA replication restart is still unclear. To understand the biochemical property and primosome assembly activity of these proteins, we expressed priA and dnaD genes which are isolated from Geobacillus stearothermophilus and Streptococcus pneumoniae and purified these proteins. We used fluorescence polarization assay to identify the protein-protein and protein-DNA interaction. The results showed that PriA carried a high affinity to DnaD and both proteins have DNA binding ability. To explain a mechanistic model of replication restart and elucidate their regulatory roles, we are trying to study the relationship between structure and function by X-ray crystallography and solved the crystal structure of DnaD at 2.5 Å resolutions by multi-wavelength anomalous diffraction. Unfortunately, we did not observe a continuous density of DnaD C-terminal region because of its flexibility. All in all, these preliminary results of initial protein structure and biochemical property give us a positive feedback for further structure determination and discussion of DNA replication restart.
關鍵字(中) ★ 蛋白質結構
★ 引發體		 關鍵字(英) ★ Protein structure
★ Primosome
論文目次 摘要 i
ABSTRACT ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 ix
一、 緒論 1
1-1 細菌中DNA複製重啟 (replication restart) 1
1-2 PriA 2
1-3 DnaD 3
1-4 嗜熱脂肪芽孢桿菌 (Geobacillus stearothermophilus) 4
1-5 肺炎鏈球菌 (Streptococcus pneumoniae) 4
二、 材料與實驗方法 5
2-1 蛋白質序列分析 5
2-1-1 多重蛋白質序列之比對 (protein sequences alignment) 5
2-2 質體建構與蛋白質表現 5
2-2-1 GstPriA 5
2-2-2 SpPriA和SNAP-SpPriA 6
2-2-3 SpDnaD、SpDnaD N端1-118和SpDnaD C端119-225 7
2-2-4 甲硒氨酸修飾 (Se-Met labelling) 之SpDnaD 7
2-3 蛋白質純化 8
2-4 螢光極化 (fluorescence polarization) 10
2-4-1 SNAP-SpPriA與SpDnaD交互作用 10
2-4-2 SpDnaD和DNA之交互作用 10
2-5 SpPriA／SpDnaD complex的size exclusion chromatography分析 11
2-6 SpPriA／SpDnaD complex的化學交聯 (cross-linking) 分析 11
2-7 蛋白質結晶 12
2-7-1 高通量篩選 12
2-7-2 GstPriA-DNA complex結晶 12
2-7-3 SpPriA-DNA complex結晶 12
2-7-4 SpDnaD與Se-Met labelling SpDnaD晶體 13
2-8 SpDnaD晶體繞射圖譜資料收集 13
三、 實驗結果 15
3-1 蛋白質序列分析 15
3-1-1 PriA 15
3-1-2 DnaD 17
3-2 蛋白質表現與純化 17
3-2-1 GstPriA 18
3-2-2 SpPriA和SNAP-SpPriA 19
3-2-3 SpDnaD、SpDnaD N端、SpDnaD C端和Se-Met SpDnaD 20
3-3 SpDnaD和SpPriA之間的交互作用 21
3-4 SpPriA和SpDnaD N端或SpDnaD C端的交互作用 21
3-5 SpPriA／SpDnaD complex的size exclusion chromatography分析 22
3-6 SpPriA／SpDnaD complex的化學交聯分析 22
3-7 SpDnaD和DNA的交互作用 23
3-8 SpDnaD與Se-Met labelling SpDnaD晶體 23
3-9 SpDnaD晶體繞射圖譜資料收集 24
3-10 SpDnaD之整體結構 25
3-11 SpDnaD結構之等電性分析 25
3-12 SpDnaD之演化保守性 26
3-13 SpDnaD結構之tetramer分析 26
四、 討論 28
五、 未來研究方向 31
5-1 GstPriA-DNA複合體結晶 31
5-2 SpPriA-DNA複合體結晶 31
5-3 SpDnaD-DNA複合體結晶 32
參考文獻 33
圖 41
表 80
附錄 83
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指導教授 蕭傳鐙 王健家(Chwan-Deng Hsiao Chien-Chia Wang) 審核日期 2019-7-26
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