大腸桿菌引子合成體蛋白PriB與單股DNA複合體之結晶結構研究

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研究生：	許哲雄
論文名稱：	大腸桿菌引子合成體蛋白PriB與單股DNA複合體之結晶結構研究 Crystal Structure of Escherichia coli primosomal protein PriB bound to single-stranded DNA
指導教授：	孫玉珠
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2005
畢業學年度：	94
語文別：	中文
論文頁數：	56
中文關鍵詞：	引物子合成體、複製、晶體結構、蛋白質
外文關鍵詞：	prib, ssb, dna replication, crystal structure, primosome, ssDNA
相關次數：	點閱：3 下載：0
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PriB，大腸桿菌replication restart primosome的組成之ㄧ，是一個複製叉 (replication fork) 受損後重新起始過程所需的蛋白。PriB 也是一個在噬菌體基因複製過程中引物合成體 (primosome)聚集所需的重要蛋白。根據過去的研究，PriB和單股DNA結合蛋白 (Single-stranded DNA binding protein，簡稱SSB)在三級結構上有很高的相似度，為了研究PriB和單股DNA的結合作用關係，我們試驗了PriB和單股DNA分別是dT15和dT30複合晶體的養成，結果分別得到解析度可達2.8和4.0的片狀晶體，兩個不同單股DNA長度的複合晶體具有相同的空間P212121。整體而言，與DNA結合後對於PriB的主要結構改變並不大，只在loop的地方有著較大的變異，尤其是L45 loop有著最顯著的不同。這個複合的結構顯示出PriB主要是利用帶正電胺基酸和單股DNA產生靜電力作用， L45 loop對於單股DNA走勢的調控扮演很重要的角色。經由此結構，我們能夠了解大腸桿菌DNA複製重新起始蛋白PriB和單股DNA分子間的作用，除此之外，這個複合結構提供一個假設的蛋白質之間作用的區域，可以用來假設PriB在引物合成體聚集過程中可能的角色。

Abstract
PriB, a component of replication restart primosome in E. coli, is a protein necessary for replication restart on the collapsed or disintegrated replication fork. PriB is also an important protein for the assembly of primosome onto ΦX174 genome DNA during replication process. Our previous work showed that PriB is structurally similar to the N-terminal DNA-binding domain of E. coli SSB. In order to investigate the single-stranded DNA binding mode of PriB, we have determined the crystal structure of PriB in complex with dT15 and dT30. These two crystals diffracted to 2.8 and 4.0 □ resolution respectively show identical crystal packing and belong to the same orthorhombic space group P212121. The ssDNA bound state of the PriB is similar to that of the apo form, with significant conformational changes only in the Loop regions, especially in the L45 loop. The structure showed that PriB binds ssDNA primarily by electrostatic interactions of positive-charged residues. L45 loop play important roles in mediating the ssDNA binding path. This structure allows us to propose a molecular basis for DNA binding of E. coli primosomal protein PriB, and furthermore, provides an implication for the putative protein interaction surface that may define the role of PriB in the assembly of primosome.

Contents

Chapter1  Introduciton
  1.1 DNA replication  -------------------------------1
  1.2 ΦX174-type primosome  --------------------------2
  1.3 Replication restart primosome of E. coli  ------3
  1.4 PriB  ------------------------------------------4
  1.5 The aim of this research  ----------------------5

Chapter2  Material and Methods
  2.1 Cloning, protein expression and purification  --7
  2.2 Crystallization  -------------------------------8
  2.3 Data collection and processing  ----------------9
  2.4 Structure determination and refinement   ------10

Chapter 3  Results
  3.1 Data collection and Space group determination  13
  3.2 Quality of the PriB-dT15 structure  -----------14
  3.3 Structure elements of PriB monomer-------------15
  3.4 Quaternary structure of PriB  -----------------16
  3.5 Arrangement of PriB in the Crystal packing  ---16
  3.6 Conformational change upon binding of PriB  ---17
  3.7 Oligo(dT) conformation  -----------------------18
  3.8 Residues involved in ssDNA binding  -----------20
  3.9 PriB-dT30 complex  ----------------------------22
  3.10 PriB-dA15 complex  ---------------------------23

Chapter 4  Discussion
  4.1 Comparison PriB with single-stranded DNA Binding
      Protein  --------------------------------------24
  4.2 Important polar residues of PriB  -------------26
  4.3 The essential role of L45 loop  ---------------27
  4.4 Implication for protein-protein interaction  --28
Figures and Figure Legends  -------------------------30
Tables  ---------------------------------------------47
Appendex  -------------------------------------------52
References  -----------------------------------------53

                                

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