進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-2907201910061300
論文名稱(中文) 探討困難梭狀芽孢桿菌DNA聚合酶I之功能與特性
論文名稱(英文) Functions and Properties of Clostridium difficile DNA Polymerase I
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 107
學期 2
出版年 108
研究生(中文) 巫祖潁
研究生(英文) Tsu-Ying Wu
學號 T36064063
學位類別 碩士
語文別 中文
論文頁數 61頁
口試委員 指導教授-陳呈堯
口試委員-黃一修
口試委員-余建泓
口試委員-阮振維
中文關鍵字 困難梭狀芽孢桿菌  DNA聚合酶I 
英文關鍵字 Clostridium difficile  DNA polymerase I 
學科別分類
中文摘要 困難梭狀芽孢桿菌是革蘭氏陽性會產生孢子的厭氧菌,是造成健康護理相關感染的主要致病源,包括抗生素治療後誘發的相關性腹瀉和偽膜性腸炎。近年來,特定核糖型困難梭狀芽孢桿菌的出現與流行,造成嚴重困難梭狀芽孢桿菌感染(CDI)的病例增加。孢子、TcdA和/或TcdB毒素都是CDI的主要的毒力因子。困難梭狀芽孢桿菌的孢子是休眠細胞,對胃的酸性環境和許多抗微生素具有高度的抗性。一旦孢子進入腸胃道,會黏附在結腸上皮細胞上,並萌發成為營養細胞。此時期的困難梭狀芽孢桿菌會分泌TcdA和/或TcdB毒素,誘導細胞發炎性趨化因子和細胞因子的產生,並造成嗜中性白血球的滲入,最終導致結腸上皮組織細胞的破壞。雖然困難梭狀芽孢桿菌的生理特性和發病機制已經廣泛地被研究,但困難梭狀芽孢桿菌本身的DNA複製合成機制尚未被探索,直至今日我們對困難梭狀芽孢桿菌的DNA聚合酶的生化功能和特性仍然一無所知。在本論文研究中,透過對蛋白質序列的比對,我們發現困難梭狀芽孢桿菌的DNA聚合酶I (CdPol I)與大腸桿菌DNA聚合酶I (EcPol I)具有高度的同源性,為了解CdPol I的生化特性,我們純化了CdPol I蛋白,並利用生物化學方法測定其活性與功能。結果顯示CdPol I含有5'→3'核酸外切酶 (5-Exo) 和5'→3'聚合酶 (DNA Pol) 酵素活性,但缺乏3'→5' 核酸外切酶或複製錯誤校對的功能。另外,我們也確認了CdPol I的5-Exo和DNA Pol活性所需的關鍵殘基。從生化測定的結果顯示,CdPol I含有DNA鏈置換、5'-flap核酸內切酶與相似於核糖核酸酶H的活性。為了瞭解CdPol I在菌內的功能,我們進一步採用ClosTron技術破壞菌中的polA基因。結果顯示,CdPol I的DNA Pol功能的破壞,造成困難梭狀芽孢桿菌的生長速率明顯地降低。然而,DNA Pol缺陷的困難梭狀芽孢桿菌的生長,在氧化壓力的條件下則呈現正常。綜合以上實驗結果,我們推斷CdPol I在困難梭狀芽孢桿菌中在DNA複製和修復中可能扮演重要的作用。
關鍵字 : 困難梭狀芽孢桿菌、DNA聚合酶I
英文摘要 SUMMARY
Clostridium difficile (C. difficile) has become a leading cause of health care-associated infections, including antibiotic-associated diarrheal disease and pseudomembranous colitis. The emergence of epidemic ribotypes of C. difficile attributes to the surge of severe cases of C. difficile infection (CDI). Although the physiology and pathogenesis of C. difficile have been extensively studied, the fundamental mechanism of DNA replication in C. difficile has not been explored. The function and requirement of DNA polymerase I in C. difficile are still unknown. To understand the biochemical features of C. difficile DNA polymerase I, designated as CdPol I in this study, we expressed, purified CdPol I, and characterized the intrinsic properties by in vitro biochemical assays. The results show that CdPol I contains a 5'→3' exonuclease (5'-Exo) and 5'→3' DNA polymerase (DNA Pol) activities, but naturally lacks a 3'→5' exonuclease, or error-proofreading, activity. Additionally, the DNA Pol domain of CdPol I has DNA strand displacement, 5'-flap endonuclease, and RNase H-like activitie. To investigate the requirement of CdPol I in vivo, the ClosTron technology was used to disrupt the polA gene in C. difficile. The disruption of CdPol I DNA Pol domain significantly reduces the growth rate of C. difficile. Taken together, our results suggest that CdPol I may play an important role in DNA replication and repair in C. difficile.

Key words: Clostridium difficile, DNA polymerase I
論文目次 中文摘要.............I
英文延伸摘要.............II
誌謝..............VI
表目錄.............XI
圖目錄..............XII
第一章 研究背景介紹...........1
一、困難梭狀芽孢桿菌...........1
二、Clostridium difficile流行病學.........2
三、Clostridium difficile傳播途徑.........2
四、Clostridium difficile致病機轉.........3
五、Clostridium difficile DNA複製蛋白........4
六、研究目的............4
第二章 材料與方法...........6
一、材料............6
(一)化學藥品...........6
(二)培養基配製...........7
(三)細菌株與表現載體..........7
(四)酵素............8
(五)目標引子之設計...........8
二、重組質體之製備..........8
(一) CdPol I 蛋白表達載體之建構........8
(二) C. difficile polA基因之定點突變........9
(三) C. difficile polA基因5'→3'外切酶區域剃除 (deletion)之建構..9
(四) CdLigA 蛋白表達載體之建構.......9
(五)聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)...10
(六) DNA 接合反應 (Ligation).........10
(七)製備勝任細胞 (Competent cells).......11
(八)勝任細胞之轉化 (Transformation)......11
三、DNA和蛋白膠體電泳........11
(一)膠體電泳 (Agarose Gel Electrophoresis)......11
(二) SDS聚丙烯醯胺膠體電泳 (SDS-PAGE)......12
(三)尿素聚丙烯醯胺膠體電泳 (Urea-PAGE)......12
四、蛋白表達與純化.........13
(一) CdPol I與其突變體之蛋白表達.......13
(二) CdLigA之蛋白表達.........13
(三)重組蛋白CdPol I之純化.........14
(四)重組蛋白CdPol I的5'→3'外切酶區域缺失之純化....15
(五)重組蛋白CdPol I突變體之純化........16
(六)重組蛋白C. difficile DNA Ligase A (CdLigA)之純化....17
五、蛋白活性之測定..........17
(一)製備雙鏈DNA或RNA-DNA底物 (substrates)....17
(二) DNA聚合酶活性分析 (Primer-Extension assay)....17
(三) 5'→3'外切酶活性分析 (Exonuclease assay)......18
(四) 3'→5'外切酶活性分析 (Exonuclease assay)......18
(五) DNA聚合酶連接酶連鎖反應分析 (DNA polymerase-ligase assay).19
六、ClosTron技術剃除C. difficile polA基因.....19
(一) C. difficile polA基因之內含子 (intron) 插入位點....19
(二) Closton plasmid之製備.........20
(三) C. difficile與Closton plasmid之共軛轉移 (Conjugate)...20
七、CdPol I聚合酶結構域對C. difficile之影響....21
(ㄧ) C. difficile DNA Pol I缺陷型之生長速率......21
(二)氧壓環境之生長速率..........21
第三章 研究結果...........22
一、C. difficile DNA聚合酶 I與E. coli DNA聚合酶I為同源蛋白..22
二、CdPol I的生化功能與特性........22
(一) CdPol I之表達與純化........22
(二) CdPol I具有DNA聚合酶和核酸外切酶活性.....23
(三) CdPol I的5'→3'核酸外切酶優選雙股DNA substrate...23
(四) CdPol I的5'→3'核酸外切酶結構域點突變之影響.....24
(五) CdPol I的5'→3'核酸外切酶具有RNase H-like活性....24
三、CdPol I在lagging-strand DNA合成中的作用.....25
(一) CdPol I具有缺口填充(gap-filling)和鏈置換合成(strand-displacement
synthesis)活性..........25
(二) CdPol I具有切口平移(nick-translation)活性.....26
(三) CdPol I具有5’-flap核酸內切酶活性.......26
四、CdLig A參與Okazaki fragment的發展.....27
(一)由CdPol I切割的5'-flap DNA產生的缺口可被CdLigA密封..27
五、CdPol I的DNA聚合酶結構域缺陷.......28
(一) C. difficile DNA聚合酶 I結構域缺陷對其生長之影響...28
(二) C. difficile DNA聚合酶 I結構域缺陷在氧化壓力下對其生長之影響.28
第四章 討論...........29
一、Asp111和Asp134殘基在CdPol I的5'-核酸外切酶結構域中的
作用.............29
二、CdPol I參與C. difficile的DNA修復作用.....30
三、在DNA複製過程中CdPol I的5'-3'外切核酸酶和DNA聚合酶之
間的相互作用...........30
四、C. difficile在不同培養基中的生長速率.....31
五、C. difficile在氧壓條件下經由損傷引起的修復.....32
第五章 參考文獻...........33
附件.............61
參考文獻 Amblar, M., et al. (2001). "Biochemical analysis of point mutations in the 5′-3′ exonuclease of DNA Polymerase I of Streptococcus pneumoniae Functional and structural implications." Journal of Biological Chemistry 276(22): 19172-19181.
2. Amblar, M. and P. López (1998). "Purification and properties of the 5′‐3′ exonuclease D190← A mutant of DNA polymerase I from Streptococcus pneumoniae." European journal of Biochemistry 252(1): 124-132.
3. Amblar, M., et al. (1998). "Purification and properties of the 5′-3′ exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing." Journal of Biotechnology 63(1): 17-27.
4. Balakrishnan, L. and R. A. Bambara (2011). "Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity." Journal of Biological Chemistry 286(9): 6865-6870.
5. Balakrishnan, L. and R. A. Bambara (2013). "Okazaki fragment metabolism." Cold Spring Harb Perspect Biol 5(2).
6. Barbut, F., P. Mastrantonio, M. Delmee, J. Brazier, E. Kuijper and I. Poxton (2007). "Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates." Clin Microbiol Infect 13(11): 1048-1057.
7. Benson, R. W., M. D. Norton, I. Lin, W. S. Du Comb and V. G. Godoy (2011). "An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents." PLoS One 6(5): e19944.
8. Bhardwaj, A., D. Ghose, K. G. Thakur and D. Dutta (2018). "Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation." PloS one 13(6): e0199559.
9. Bornhorst, J. A. and J. J. Falke (2000). "Purification of proteins using polyhistidine affinity tags." Methods in Enzymology, Elsevier. 326: 245-254.
10. Briggs, G. S., W. K. Smits and P. Soultanas (2012). "Chromosomal replication initiation machinery of low-G+ C-content Firmicutes." Journal of Bacteriology 194(19): 5162-5170.
11. Cartman, S. T. and N. P. Minton (2010). "A mariner-based transposon system for in vivo random mutagenesis of Clostridium difficile." Appl. Environ. Microbiol. 76(4): 1103-1109.
12. Cerritelli, S. M. and R. J. Crouch (2009). "Ribonuclease H: the enzymes in eukaryotes." The FEBS Journal 276(6): 1494-1505.
13. Clements, A. C., R. J. Magalhaes, A. J. Tatem, D. L. Paterson and T. V. Riley (2010). "Clostridium difficile PCR ribotype 027: assessing the risks of further worldwide spread." Lancet Infect Dis 10(6): 395-404.
14. Curry, S. R., C. A. Muto, J. L. Schlackman, A. W. Pasculle, K. A. Shutt, J. W. Marsh and L. H. Harrison (2013). "Use of multilocus variable number of tandem repeats analysis genotyping to determine the role of asymptomatic carriers in Clostridium difficile transmission." Clin Infect Dis 57(8): 1094-1102.
15. de Saro, F. J. L. and M. O'Donnell (2001). "Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I." Proceedings of the National Academy of Sciences 98(15): 8376-8380.
16. Dervyn, E., C. Suski, R. Daniel, C. Bruand, J. Chapuis, J. Errington, L. Jannière and S. D. Ehrlich (2001). "Two essential DNA polymerases at the bacterial replication fork." Science 294(5547): 1716-1719.
17. Díaz, A., et al. (1992). "The 5′ to 3′ exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae." Molecular Microbiology 6(20): 3009-3019.
18. Edwards, A. N. and S. M. McBride (2016). Isolating and purifying Clostridium difficile spores. Clostridium difficile, Springer: 117-128.
19. García-Ortíz, M.-V., et al. (2011). "Unexpected role for Helicobacter pylori DNA polymerase I as a source of genetic variability." PLoS Genetics 7(6): e1002152.
20. Garg, P., C. M. Stith, N. Sabouri, E. Johansson and P. M. Burgers (2004). "Idling by DNA polymerase δ maintains a ligatable nick during lagging-strand DNA replication." Genes & Development 18(22): 2764-2773.
21. George, R. H., et al. (1978). "Identification of Clostridium difficile as a cause of pseudomembranous colitis." Br Med J 1(6114): 695.
22. Gerding, D. N., S. Johnson, M. Rupnik and K. Aktories (2014). "Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance." Gut Microbes 5(1): 15-27.
23. Goorhuis, A., S. B. Debast, L. A. van Leengoed, C. Harmanus, D. W. Notermans, A. A. Bergwerff and E. J. Kuijper (2008). "Clostridium difficile PCR ribotype 078: an emerging strain in humans and in pigs?" J Clin Microbiol 46(3): 1157; author reply 1158.
24. Guarner, F. (2006). "Enteric flora in health and disease." Digestion 73 Suppl 1: 5-12.
25. Gutman, P. D. and K. W. Minton (1993). "Conserved sites in the 5'-3' exonuclease domain of Escherichia coli DNA polymerase." Nucleic Acids Research 21(18): 4406.
26. Hall, I. C. and E. O'Toole (1935). "Intestinal flora in new-born infants: with a description of a new pathogenic anaerobe, Bacillus difficilis." JAMA Pediatrics 49(2): 390-402.
27. Helmrich, A., et al. (2011). "Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes." Molecular Cell 44(6): 966-977.
28. Hensgens, M. P., et al. (2012). "Clostridium difficile infection in the community: a zoonotic disease?" Clin Microbiol Infect 18(7): 635-645.
29. Johanesen, P. A., et al. (2015). "Disruption of the gut microbiome: Clostridium difficile infection and the threat of antibiotic resistance." Genes (Basel) 6(4): 1347-1360.
30. Johnson, S. (2009). "Recurrent Clostridium difficile infection: a review of risk factors, treatments, and outcomes." J Infect 58(6): 403-410.
31. Karasawa, T., S. Ikoma, K. Yamakawa and S. Nakamura (1995). "A defined growth medium for Clostridium difficile." Microbiology 141(2): 371-375.
32. Keel, K., J. S. Brazier, K. W. Post, S. Weese and J. G. Songer (2007). "Prevalence of PCR ribotypes among Clostridium difficile isolates from pigs, calves, and other species." J Clin Microbiol 45(6): 1963-1964.
33. Kornberg, A. and T. A. Baker (1992). DNA Replication, W.H. Freeman.
34. Larson, H. E., et al. (1978). "Clostridium difficile and the aetiology of pseudomembranous colitis." Lancet 1(8073): 1063-1066.
35. Lawley, T. D., et al. (2009). "Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores." J Bacteriol 191(17): 5377-5386.
36. Lundquist, R. C. and B. M. Olivera (1982). "Transient generation of displaced single-stranded DNA during nick translation." Cell 31(1): 53-60
37. Lyamichev, V., M. A. D. Brow, V. E. Varvel and J. E. Dahlberg (1999). "Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain." Proceedings of the National Academy of Sciences 96(11): 6143-6148.
38. McDonald, L. C., D. N. Gerding, S. Johnson, J. S. Bakken, K. C. Carroll, S. E. Coffin, E. R. Dubberke, K. W. Garey, C. V. Gould, C. Kelly, V. Loo, J. Shaklee Sammons, T. J. Sandora and M. H. Wilcox (2018). "Clinical practice guidelines for Clostridium difficile Infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA)." Clin Infect Dis 66(7): e1-e48.
39. Mizrahi, V. and P. Huberts (1996). "Deoxy-and dideoxynucleotide discrimination and identification of critical 5′ nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis." Nucleic Acids Research 24(24): 4845-4852.
40. Neumann-Schaal, M., J. D. Hofmann, S. E. Will and D. Schomburg (2015). "Time-resolved amino acid uptake of Clostridium difficile 630Δerm and concomitant fermentation product and toxin formation." BMC Microbiology 15(1): 281.
41. Pandey, P., K. F. Tarique, M. Mazumder, S. A. A. Rehman and S. Gourinath (2016). "Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori." Scientific Reports 6: 31181.
42. Paredes-Sabja, D. and M. R. Sarker (2012). "Adherence of Clostridium difficile spores to Caco-2 cells in culture." J Med Microbiol 61(Pt 9): 1208-1218.
43. Pettit, L. J., H. P. Browne, L. Yu, W. K. Smits, R. P. Fagan, L. Barquist, M. J. Martin, D. Goulding, S. H. Duncan, H. J. Flint, G. Dougan, J. S. Choudhary and T. D. Lawley (2014). "Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism." BMC Genomics 15: 160.
44. Poutanen, S. M. and A. E. Simor (2004). "Clostridium difficile-associated diarrhea in adults." CMAJ 171(1): 51-58.
45. Roos, W. P. and B. Kaina (2013). "DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis." Cancer Letters 332(2): 237-248.
46. Rupnik, M., M. H. Wilcox and D. N. Gerding (2009). "Clostridium difficile infection: new developments in epidemiology and pathogenesis." Nat Rev Microbiol 7(7): 526-536.
47. Sanyal, G. and P. Doig (2012). "Bacterial DNA replication enzymes as targets for antibacterial drug discovery." Expert Opin Drug Discov 7(4): 327-339.
48. Sayers, J. R. and F. Eckstein (1991). "A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease." Nucleic Acids Research 19(15): 4127-4132.
49. Skourti-Stathaki, K. and N. J. Proudfoot (2014). "A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression." Genes & Development 28(13): 1384-1396.
50. Smits, W. K., D. Lyras, D. B. Lacy, M. H. Wilcox and E. J. Kuijper (2016). "Clostridium difficile infection." Nat Rev Dis Primers 2: 16020.
51. Sun, X., T. Savidge and H. Feng (2010). "The enterotoxicity of Clostridium difficile toxins." Toxins (Basel) 2(7): 1848-1880.
52. Thrall, E. S., J. E. Kath, S. Chang and J. J. Loparo (2017). "Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage." Nature Communications 8(1): 2170.
53. Uson, M. L., S. Ghosh and S. Shuman (2017). "The DNA repair repertoire of Mycobacterium smegmatis FenA includes the incision of DNA 5′ flaps and the removal of 5′ adenylylated products of aborted nick ligation." Journal of Bacteriology 199(17): e00304-00317.
54. Voth, D. E. and J. D. Ballard (2005). "Clostridium difficile toxins: mechanism of action and role in disease." Clin Microbiol Rev 18(2): 247-263.
55. Xu, Y., V. Derbyshire, K. Ng, X. C. Sun, N. D. Grindley and C. M. Joyce (1997). "Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli." Journal of Molecular Biology 268(2): 284-302.
56. Xu, Y., O. Potapova, A. E. Leschziner, N. D. Grindley and C. M. Joyce (2001). "Contacts between the 5′ nuclease of DNA polymerase I and its DNA substrate." Journal of Biological Chemistry 276(32): 30167-30177.
57. Yang, W. and Y. Gao (2018). "Translesion and repair DNA polymerases: Diverse structure and mechanism." Annual Review of Biochemistry 87: 239-261.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2024-07-30起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2024-07-30起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw