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研究生：	鄭伊雯 Cheng, Yi-Wen
論文名稱：	探討胃幽門螺旋桿菌DNA聚合酶I之生化特性 Biochemical characterization of Helicobacter pylori DNA polymerase I
指導教授：	陳呈堯 Chen, Cheng-Yao
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	75
中文關鍵詞：	胃幽門氏桿菌、DNA聚合酶I 、岡崎片段、flap核酸內切酶、核糖核酸酶H
外文關鍵詞：	Helicobacter pylori, DNA polymerase I, Okazaki fragment, Flap endonuclease, RNase H
相關次數：	點閱：51 下載：1
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胃幽門氏桿菌是常見的人類致病菌之一，全球約有一半以上的人口受到胃幽門氏桿菌的感染，此致病菌造成慢性胃炎、胃潰瘍、甚至胃腺癌。先前的研究指出胃幽門氏桿菌的DNA聚合酶I，本研究簡稱HpyPol I，可以提升此細菌體內基因體的突變率，而這也許提供了胃幽門氏桿菌一個得以生存且附著在我們腸胃道黏膜上的生長優勢。藉由基因剔除的方式干擾HpyPol I的功能會導致該細菌的死亡，顯示出HpyPol I對胃幽門氏桿菌的生存與生長是不可或缺的，然而目前對於HpyPol I的生化功能及特性仍是未知的。透過蛋白質序列的比對，我們發現HpyPol I與A族的大腸桿菌DNA聚合酶I (EcoPol I) 互為同源關係，但其缺少3端往5端的核酸外切酶的功能，或是校正DNA合成時產生錯誤的能力。在本論文研究中，我們證明了HpyPol I可以替補EcoPol I在JS200大腸桿菌中的功能，此一菌株帶有一突變的PolI DNA聚合酶 (EcoPolI) 等位基因而使其對溫度產生敏感性，當細菌生長在37度C時，對溫度敏感的EcoPolI會失去活性。根據此一研究結果我們推測HpyPol I在細菌體內DNA複製的過程中可能扮演與EcoPol I相似的角色。為了瞭解HpyPol I在DNA複製中的功能與特性，我們表達且純化了HpyPol I及其5端核酸外切酶上帶有突變的蛋白。在DNA引子延伸的實驗中，HpyPol I表現出強烈的DNA鏈置換、5端flap核酸內切酶與相似於核糖核酸酶H的活性。HpyPol I能在DNA合成中置換下游的DNA與RNA鏈，且可於單股DNA與雙股DMA間的會合處切除因置換所產生的單股DNA或RNA鏈。HpyPol I的5端核酸外切酶可有效的在具平滑末端的DNA-DNA或RNA-RNA結構進行降解。將5端核酸外切酶結構域上可能為與金屬離子契合的天門冬胺酸殘基進行突變(D121A與D141A)，會大幅地去除其5端核酸外切酶、5端flap核酸內切酶及相似於核糖核酸酶H的酵素活性。此外，我們提出在HpyPol I中，聚合酶結構區域與5端核酸外切酶結構區域之間的轉換機制是透過分子內直接轉換的途徑。綜合所有實驗結果，我們推測HpyPol I可能對胃幽門氏桿菌體內基因體複製中岡崎片段的成熟過程中，扮演一個去除RNA引子的重要角色。

Helicobacter pylori (H. pylori) is one of the most common human pathogens on the earth. More than half of the world’s population were/are infected by H. pylori, which lead to chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Previous studies have reported that DNA polymerase I of H. pylori, designated as HpyPol I in this study, contributes to an elevated genome mutation rate, which may provide growth advantages during H. pylori colonization to the mucosa of upper gastrointestinal tract. The disruption of HpyPol I function by the target gene deletion results in the death of bacteria. Although HpyPol I is essential for the growth and viability of H. pylori, its functions and properties are still unclear. HpyPol I is homologous to E. coli DNA polymerase I (EcoPol I) and belongs to the A-family DNA polymerases. Unlike EcoPol I, HpyPol lacks a functional 3’→5’ exonuclease or error-proofreading activity. In this thesis, we demonstrated that HpyPol I can functionally substitute for EcoPol I in the E. coli JS200 cell, which harbors a temperature-sensitive DNA polymerase I allele (pol Its), and may play a similar role in vivo. To understand the biochemical properties of HpyPol I, we purified recombinant HpyPol I and its 5’-3’ exonuclease (5’-Exo) mutants. In the in vitro experiments, HpyPol I shows a robust DNA strand-displacement synthesis, 5’-flap endonuclease, and RNase H-like activity. HpyPol I can displace and cleave the downstream DNA or RNA primer during DNA synthesis at the junction between the single-stranded and duplex DNA/RNA region. The 5’-Exo function of HpyPol I can also efficiently degrade DNA or RNA from a blunt-end, duplex DNA-DNA, or RNA-DNA hybrid. Mutations at the putative, metal-chelating aspartate residues (D121A and D141A) of 5’-Exo domain greatly abolish the 5’-Exo, 5’-flap endonuclease, and RNase H-like activities. Finally, we showed that the switching pathway between DNA polymerase and 5’-Exo domain is through an intramolecular pathway in HpyPol I. Taken together, our results suggest that HpyPol I may play an important role in the maturation of Okazaki fragments during the genome replication of H. pylori.

中文摘要……………………………………………………………………I
Abstract…………………………………………………………………III
誌 謝 …………………………………………………………………………V
TABLE OF CONTENTS……………………………………………………VI
LIST OF TABLE………………………………………………………………X
LIST OF FIGURE……………………………………………………………XI
INTRODUCTION………………………………………………………………1
1 The History and Prevalence of Helicobacter pylori……………………………1
2 The High Genetic Diversity of Helicobacter pylori……………………………2
3 The DNA Replication of Helicobacter pylori…………………………………3
4 The DNA Polymerase I of Helicobacter pylori………………………………4
5 The Aims of Current Studies…………………………………………4
MATERILS AND METHODS…………………………………………………6
1. Materials…………………………………………………………………………6
1.1. Chemicals…………………………………………………………………6 
1.2. Bacterial Media……………………………………………………………6
1.3. Bacterial Strains and Plasmids…………………………………6
1.4. Enzymes…………………………………………………………………7
1.5. Oligonucleotides……………………………………………………………7
2. Cloning Procedures……………………………………………………………7
2.1 Preparation of Recombinant Plasmids…………………………………………7
2.1.1. Construction of Recombinant Plasmids…………………………7
2.1.2. Construction of HpyPol I Protein Expression Vector…………8
2.1.3. Site-Directed Mutagensis of H. pylori polA gene………………8
2.1.4. Construction of HpyLigA Protein Expression Vector…………8
2.2. Polymerase Chain Reaction (PCR)……………………….9
2.3. DNA Ligation……………………………………….9
2.4. Preparation of Chemical Competent Cells…………………9
2.5. Transformation of Competent Cells…………………………10
3. DNA and Protein Gel Electrophoresis………………….………..10
3.1. Agarose Gel Electrophoresis………………………………….10
3.2. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)...11
3.3. Urea-Polyacrylamide Sequencing Gel Electrophoresis (Urea-PAGE)…..11
4. Protein Expression and Purification……………………………………12
4.1. Protein Expression of HpyPol I and its Derivatives……………………12
4.2. Protein Expression of HpyLigA…………………………….12
4.3. Purification of Recombinant HpyPol I and its Mutants…………13
4.4. Purification of Recombinant H. pylori DNA Ligase A (HpyLigA)……...14
5. Genetic Complementation and Enzymatic Assays…………………15
5.1. Genetic Complementation Assay………………………...…………15
5.2. Preparation of Duplex DNA or RNA-DNA Substrates………16
5.3. DNA Polymerase Activity Assay (Primer-Extension Assay)……….16
5.4. 5’→3’ Exonuclease Assay (Exonucleolytic Assay…………..17
5.5. DNA Polymerase-Ligase Assay………………………..17
5.6. Optimization of Heparin Concentration to Trap the Free DNA Polymerase…….17
5.7. Heparin Trap Assay to Monitor the Switch Pathway Between DNA Polymerase and Exonuclease Domain………..18
RESULTS……………………………………………….20
1 HpyPol I Functionally Substitutes for E. coli DNA Polymerase I In Vivo ………20
2 The Biochemical Functions and Properties of the 5’→3’ Exonuclease of HpyPol I..21
2.1. Expression and Purification of HpyPol I………………21
2.2. HpyPol I Contains both DNA Polymerase and Exonuclease Activities…….21
2.3. The 5’→3’ Exonuclease of HpyPol I Prefers a Double Strand DNA Substrate…22
2.4. The 5’→3’ Exonuclease of HpyPol I Contains a RNase H-like Activity….…22
2.5. The Mutational Effects on 5’→3’ Exonuclase Domain of HpyPol I………22
3 The Role of HpyPol I in the Lagging Strand DNA Synthesis………23
3.1. HpyPol I has a Nick-Translation Activity…………24
3.2. HpyPol I has a Gap-Filling and Strand-Displacement Synthesis Activity…….…24
3.3. HpyPol I has a Flap Endonuclease Activity……25
4 The Involvement of HpyLig A in the Maturation of Okazaki Fragments………..26
4.1. A DNA Nick Resulting From the 5’ Flap DNA Cleavage by HpyPol I Can Be Sealed by HpyLigA……………………….26
5 The DNA Polymerase (Pol) and Exonuclease (Exo) Domain Switch of HpyPol I.….27
5.1. The Functional Switch of HpyPol I Pol-Exo Domain is Through a Direct Intra Molecular Exchange….……...27
DISCUSSION………………………………………………………….29
1 The Role of Asp121 and Asp141 in the 5’ Exonuclease Domain of HpyPol I…….29
2 The Functions of HpyPol I in the H. pylori DNA Repair……………...…30
3 The Idling of HpyPol I During the H. pylori Genome Replication…………….31
4 Maturation of Okazaki Fragments During the H. pylori Genome Replication…..32 
5 Summary……………………………………………………………32
REFERENCES……………………………………………...33
TABLES………………………………………………..…….40
Table 1. Oligonucleotides Used in the Biochemical Assays…...40
Table 2. Oligonucleotides Used in DNA Cloning……..….41
FIGURES……………………………………………….42
APPENDIX…………………………………………74
Appendix 1. The pET15b-HpyPol I Map……….…...........74
Appendix 2. The Protein Sequence Alignment of 5’→3’ Exonuclease Domain of E. coli and H. pylori DNA Polymerase I……………...................75
                                    

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