染色體分配系統是在細胞週期中確保染色體能正確分配到子細胞的系統。真核生物及細菌的分配系統已被廣泛地研究，但目前對於古細菌物種分離染色體的機制所知尚少。過去的研究提出古細菌硫磺礦硫化葉菌(Sulfolobus solfataricus)的染色體分配系統(SegAB系統)1，其由兩種蛋白質SegA、SegB以及SegB的DNA結合位(segregation site, segS)組成。SegA是一種ATP水解酶，並具有形成聚合體的特性。SegB是一種DNA結合蛋白，能夠專一性地結合於segS。研究指出SegAB系統的染色體分離機制可能與ParABS系統相似，但仍有待研究。
本篇論文的研究目的是探討SegAB系統的機制，並著重於分析SegB的生物功能及SegB與segS的複合物分子結構。我們預測SegB的二級結構含有六段α螺旋及一段β折疊，並推測其可能利用Ribbon-Helix-Helix結構體或Helix-Turn-Helix結構體結合DNA。我們利用電泳遷移率變動分析(EMSA)驗證SegB與segS的專一性，並確認了SegB與不同長度segS的結合能力差異。實驗中利用X光晶體繞射方法解析SegB和segS的複合物結構。我們利用蒸氣擴散法獲得SegB-SE19複合物晶體，其分子空間群為六方晶系(hexagonal, P3)。由Matthews coefficient推測其不對稱單元(asymmetric unit)由三個SegB-SE19複合物組成。我們也獲得突變蛋白SegBΔ22-SE19複合物晶體。由於該晶體為多晶，無法決定空間群分類。目前仍需優化晶體培養條件，以獲得質量好的SegB-segS複合物晶體，並收集完整的X光繞射數據。

Chromosome partition system ensures an accurate distribution of the genetic material to the daughter cells. The partition systems of eukaryotes and bacteria have been extensively studied, but the partition system of archaea is poorly understood. In the past research, the chromosome segregation system (SegAB system) of Sulfolobus solfataricus was proposed1. The SegAB system is composed of two proteins, SegA, SegB and a specific DNA binding site (segS). SegA is an ATPase and is able to assemble into polymers. SegB is a DNA binding protein and binds to segS specifically. The SegAB system might separate the chromosomes by the same way as the ParABS system. The mechanism of the SegAB system remains to be studied.
The aims are to study the mechanism of the SegAB system, and we focused on analyzing the biological functions of SegB and solving the structure of SegB-segS complex. According to the structural predictions, SegB contains six α-helix and one β-sheet, and we assumed SegB might interact with segS through Ribbon-Helix-Helix motif or Helix-Turn-Helix motif. We analyzed the binding affinity between SegB and segS by the electrophoretic mobility shift assay. We used protein crystallography method to solve the crystal structure of SegB-segS complex, and obtained SegB-SE19 complex crystal by vapor diffusion method, which belongs to P3 space group. The crystal could contain three SegB-SE19 complexes in one asymmetry unit. We also obtained SegBΔ22-SE19 complex crystal, which formed multi-crystal, that the space group could not be decided. We need to optimize the crystallization condition to improve SegB-segS complex crystal and to get complete X-ray diffraction data.

目錄 I
摘要 III
Abstract IV
誌謝 V
第一章 簡介 1
1.1 硫磺礦硫化葉菌(Sulfolobus solfataricus) 1
1.2 基因組分離(Genome segregation) 2
1.2.1 ParABS系統 2
1.2.2 SegAB系統 3
1.3 SsoSegB 4
1.4 SegB的DNA結合位segS 5
第二章 材料與方法 6
2.1 SegB蛋白質表現與純化 6
2.1.1 SegB基因載體 6
2.1.2 SegB蛋白表現 6
2.1.3 Ni2+-NTA管柱純化 7
2.1.4離子交換管柱純化 7
2.1.5突變蛋白SegBΔ22製備 8
2.2 segS DNA設計與製備 8
2.3 SegB-segS複合物製備 9
2.4 SegB與DNA的結合分析 9
2.4.1電泳遷移率變動分析(Electrophoretic mobility shift assay, EMSA) 9
2.4.2非變性聚丙烯醯胺凝膠電泳(Native PAGE) 10
2.5分子篩選層析(size exclusion chromatography, SEC) 10
2.6晶體培養(Crystallization) 10
第三章 結果與討論 12
3.1 SegB蛋白特性 12
3.1.1 SegB的胺基酸特性 12
3.1.2 SegB的序列比對結果 12
3.1.3 SegB的二級結構預測 13
3.1.4 SegB與DNA結合預測 13
3.2 SegB的表現與純化 14
3.3不同酸鹼值中的SegB 15
3.3.1 SegB的狀態分析 15
3.3.2 SegB的穩定性分析 16
3.4 SegB-segS複合物 17
3.4.1 SegB與不同長度segS DNA的結合能力 17
3.4.2兩種類型的SegB-segS複合物 17
3.4.3 分子篩選層析結果 18
3.5 SegB-segS複合物晶體 19
3.6 SegB-SE19複合物晶體繞射數據 20
3.7 SegBΔ22-SE19複合物晶體繞射數據 21
第四章 總結 22
表格與圖片 24
參考資料 45

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