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論文中文名稱:複製中鏈黴菌DNA端粒的引子酶辨認序列之研究 [以論文名稱查詢館藏系統]
論文英文名稱:To find the primase recognition site at the telomere of Streptomyces [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:102
出版年度:103
中文姓名:吳尚芳
英文姓名:Shang-Fang Wu
研究生學號:101688002
學位類別:碩士
語文別:中文
口試日期:2014-07-22
論文頁數:63
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金
中文關鍵詞:鏈黴菌端粒複製引子酶辨認序列
英文關鍵詞:Streptomycestelomere replicationprimase recognition sequence
論文中文摘要:鏈黴菌是帶有線型染色體及質體的革蘭氏陽性菌,其線型DNA的複製是由中間開始向兩端同時進行雙股複製,複製到末端時其延遲股(lagging strand)無法複製到最末段,在不同的端粒(telomere)序列會留下不同長度的單股間隙,是為端粒複製中間產物,然後此中間產物的單股DNA部分預期會依其豐富的廻紋序列自動形成穩定的二級結構供Tap蛋白所辨識結合,再藉由Tap與TP(末端蛋白)及DNA聚合酶的互相作用,將後兩者帶至這中間產物上,然後DNA聚合酶以TP作為複製起始所需的引子、此中間產物為模板,將其單股DNA片段複製完成。所以這端粒複製中間產物的出現,應該就是端粒複製的起始訊號。雖這中間產物在不同端粒序列上所出現的單股DNA長度的不同,推測除了決定岡崎片段位置的引子酶(primase)的辨識序列在不同的端粒序列上的位置不同外,其豐富的廻紋序列的位置可能也有關。為了研究這個問題,藉由找尋最後一個岡崎片段出現的位置來得知鏈黴菌的引子酶辨識序列是首需解決的問題,並可進一步探討其位置對端粒複製的影響。本論文以帶有S. lividans染色體端粒片段的線型質體pLUS970L做為研究對象,藉由在端粒序列上的各種突變來觀察其延遲股上最後一個岡崎片段位置是否受影響,來找尋其引子酶辨識序列的位置。本實驗室的先前研究,已將其位置縮小到10個核苷酸之間,而我繼續縮小範圍到五個核苷酸之間。此外我也藉由在線型質體上構築相同長度的不同端粒序列,比較其岡崎片段位置的不同位置來找是否有保守序列來幫助尋引子酶辨識序列,結果發現有CGGSCS的保守序列,值得作為後續研究的起始目標。而比較這些不同端粒上最後岡崎片段的位置與廻紋序列區域的關係,發現其位置都是落在廻紋序列區域的外面,可能暗示廻紋序列可能與最後一個岡崎片段出現的位置也有關係。
論文英文摘要:Streptomyces is gram-positive bacteria with linear chromosome/plasmid. Replication of linear DNA in Streptomyces is initiated from an internal origin and proceeds bidirectionally towards the ends. In this mechanism, lagging strand leaves a single-strand gap at 3’ end. This gap, known as a telomeric replication intermediate, is rich in palindromes and is expected to fold into stable secondary structure for Tap (telomere-associated protein) binding. TP (terminal protein) and DNA polymerase are recruited to this intermediate by interaction with Tap, then the DNA polymerase employees TP as primer and the intermediate as template to complete the single-strand gap. Thus, the appearance of replication intermediate might be considered as an initiating signal of telomere replication. The length of replication intermediates vary in different telomeres. We hypothesized that the gap is not only decided by primase recognition site responsible for the last Okazaki fragment, the pattern and locations of palindromes might also take part in it. Localization of the last primase recognition sequence will be the first step to further reveal the mechanism of Streptomyce telomeric replication and how the position of primase recognition sequence can affect telomere replication.
Our lab’s previous studies used linear plasmid pLUS970L with S. lividans telomeres as research material, several telomere mutations were generated to examine the position of last Okazaki fragment in telomere. By this strategy, the last primase recognition sequence had narrowed down in 10 base pairs. In this study, I continued to narrow it to 5 base pairs. I also tried to find primase recognition sequence by comparing the conserved sequences in the upstream of last Okazaki fragment in pLUS970L derivatives with the same length of different telomeres. I found that the conserve region contenting CGGSCS can be a start point of further studies. Comparing the position of the last Okazaki fragment and the distribution of palindromes, I found that the last Okazaki fragment always located outside the clustered palindromes. It suggest that the palindromes might also affect the position of last Okazaki fragment.
論文目次:中文摘要 i
英文摘要 iii
誌 謝 v
目錄 vi
圖目錄 viii
第一章 緒論 1
1.1鏈黴菌的簡介 1
1.2鏈黴菌線形DNA複製 2
1.3引子酶與解旋酶的互相作用 6
1.4本論文內容 10
第二章 材料與方法 11
2.1菌種及質體 11
2.2藥品、酵素 14
2.3培養基及緩衝溶液 14
2.4菌種之儲存 14
2.5大腸桿菌之轉型 15
2.6大腸桿菌質體之分離與純化 15
2.7鏈黴菌原生質體的製備與轉型 15
2.8鏈黴菌基因組DNA的分離與純化 15
2.9限制酶酵素、T4連接酵素及各種kit的使用方式 16
2.10聚合酶連鎖反應PCR(polymerase chain reaction) 16
2.11南方點墨法(Southern blot hybridization) 16
2.12鏈黴菌線狀微型質體的構築 16
2.13鏈黴菌生長曲線的測試 17
2.14收下在對數中期生長的鏈黴菌 18
2.15純化大量鏈黴菌總DNA 18
2.16以BND cellulose純化末端單股間隙 19
2.17 End junction序列檢測方法 20
2.18計算端粒複製中間產物單股DNA片段長度 22
第三章 實驗結果 23
3.1化複製中的pLUS970L的條件最佳化 .......23
3.2從末端第276-285 bp區間繼續縮小引子酶的可能範圍 25
3.3 插入32 bp的方法來尋找位於末端第275-285之間位置的引子辨認序列 34
3.4以端粒到限制酶BclI同樣長度,置換不同線形DNA末端序列來比較可能之辨認序列 37
第四章 討論 41
參考文獻 47
附錄
A. Media and buffer 52
B. E. coli Competent cell preparation and transformation 54
C. Plasmid isolation from E. coli 57
D. Isolation the total DNA of Streptomyces 58
E. Polymerase Chain Reaction 59
F. Southern hybridization 60
論文參考文獻:1. Bao, K., and Cohen, S.N. (2001). Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. Genes & development 15, 1518-1527.
2. Bao, K., and Cohen, S.N. (2003). Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication. Genes & development 17, 774-785.
3. Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., et al. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141-147.
4. Berg JM, T.J., Stryer L, Clarke ND (2002). DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites.
5. Birch, A., Hausler, A., and Hutter, R. (1990). Genome rearrangement and genetic instability in Streptomyces spp. Journal of bacteriology 172, 4138-4142.
6. Bird, L.E., Pan, H., Soultanas, P., and Wigley, D.B. (2000). Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry 39, 171-182.
7. Biswas, E.E., and Biswas, S.B. (1999). Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization. Biochemistry 38, 10919-10928.
8. Berg, J.M., Tymoczko,J.L., Stryer, L., Clarke, N.D., Biochemistry. W.H. Freeman and Company, 2002, Chapter 27, Section 4
9. Bouche, J.P., Zechel, K., and Kornberg, A. (1975). dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. The Journal of biological chemistry 250, 5995-6001.
10. Challberg, M.D., Desiderio, S.V., and Kelly, T.J., Jr. (1980). Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands. Proceedings of the National Academy of Sciences of the United States of America 77, 5105-5109.
11. Chang, P.C., and Cohen, S.N. (1994). Bidirectional replication from an internal origin in a linear streptomyces plasmid. Science 265, 952-954.
12. Corn, J.E., and Berger, J.M. (2006). Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions. Nucleic acids research 34, 4082-4088.
13. Chaconas,G. and Chen,C.W., The Bacterial Chromosome., American Society for Microbiology,Washington, DC, pp. 525–539.
14. Clark,D.P., Elsevier Pet Ltd, 2005, p121-122
15. Fischer, G., Decaris, B., and Leblond, P. (1997). Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA. Journal of bacteriology 179, 4553-4558.
16. Frick, D.N., and Richardson, C.C. (1999). Interaction of bacteriophage T7 gene 4 primase with its template recognition site. The Journal of biological chemistry 274, 35889-35898.
17. Frick, D.N., and Richardson, C.C. (2001). DNA primases. Annual review of biochemistry 70, 39-80.
18. Heller, R.C., and Marians, K.J. (2006). Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557-562.
19. Hopwood, D.A. (2006). Soil to genomics: the Streptomyces chromosome. Annual review of genetics 40, 1-23.
20. Huang, C.H., Lin, Y.S., Yang, Y.L., Huang, S.W., and Chen, C.W. (1998). The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures. Molecular microbiology 28, 905-916.
21. Huang, C.H., Tsai, H.H., Tsay, Y.G., Chien, Y.N., Wang, S.L., Cheng, M.Y., Ke, C.H., and Chen, C.W. (2007). The telomere system of the Streptomyces linear plasmid SCP1 represents a novel class. Molecular microbiology 63, 1710-1718.
22. Koepsell, S.A., Larson, M.A., Griep, M.A., and Hinrichs, S.H. (2006). Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity. Journal of bacteriology 188, 4673-4680.
23. Kuchta, R.D., and Stengel, G. (2010). Mechanism and evolution of DNA primases. Biochimica et biophysica acta 1804, 1180-1189.
24. Kusakabe, T., Hine, A.V., Hyberts, S.G., and Richardson, C.C. (1999). The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition. Proceedings of the National Academy of Sciences of the United States of America 96, 4295-4300.
25. Krebs, J.E., Goldstein, E.S., Kilpatrick, S.T., Lewin’s Essential GENES, America:Jones and Bartlett Publishers, 2009, p.422-443
26. Kornberg,A. and Baker,T., DNA Replication, San Francisco, 1992
27. Larson, M.A., Bressani, R., Sayood, K., Corn, J.E., Berger, J.M., Griep, M.A., and Hinrichs, S.H. (2008). Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines. Nucleic acids research 36, 5260-5269.
28. Larson, M.A., Griep, M.A., Bressani, R., Chintakayala, K., Soultanas, P., and Hinrichs, S.H. (2010). Class-specific restrictions define primase interactions with DNA template and replicative helicase. Nucleic acids research 38, 7167-7178.
29. Lee, S.J., Zhu, B., Hamdan, S.M., and Richardson, C.C. (2010). Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic acids research 38, 4372-4383.
30. Lin, Y.S., Kieser, H.M., Hopwood, D.A., and Chen, C.W. (1993). The chromosomal DNA of Streptomyces lividans 66 is linear. Molecular microbiology 10, 923-933.
31. Lu, Y.B., Ratnakar, P.V., Mohanty, B.K., and Bastia, D. (1996). Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA. Proceedings of the National Academy of Sciences of the United States of America 93, 12902-12907.
32. Mitkova, A.V., Khopde, S.M., and Biswas, S.B. (2003). Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis. The Journal of biological chemistry 278, 52253-52261.
33. Meijer, W.J., Lewis, P.J., Errington, J., and Salas, M. (2000). Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7. The EMBO journal 19, 4182-4190.
34. Norcum, M.T., Warrington, J.A., Spiering, M.M., Ishmael, F.T., Trakselis, M.A., and Benkovic, S.J. (2005). Architecture of the bacteriophage T4 primosome: electron microscopy studies of helicase (gp41) and primase (gp61). Proceedings of the National Academy of Sciences of the United States of America 102, 3623-3626.
35. Oakley, A.J., Loscha, K.V., Schaeffer, P.M., Liepinsh, E., Pintacuda, G., Wilce, M.C., Otting, G., and Dixon, N.E. (2005). Crystal and solution structures of the helicase-binding domain of Escherichia coli primase. The Journal of biological chemistry 280, 11495-11504.
36. Pan, H., and Wigley, D.B. (2000). Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase. Structure 8, 231-239.
37. Penalva, M.A., and Salas, M. (1982). Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP. Proceedings of the National Academy of Sciences of the United States of America 79, 5522-5526.
38. Rowen, L., and Kornberg, A. (1978). Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. The Journal of biological chemistry 253, 758-764.
39. Sakaguchi, K. (1990). Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses. Microbiological reviews 54, 66-74.
40. Shih, M., Watabe, K., and Ito, J. (1982). In vitro complex formation between bacteriophage phi 29 terminal protein and deoxynucleotide. Biochemical and biophysical research communications 105, 1031-1036.
41. Spatz, K., Kohn, H., and Redenbach, M. (2002). Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2. FEMS microbiology letters 213, 87-92.
42. Sun, W., and Godson, G.N. (1996). Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex. Journal of bacteriology 178, 6701-6705.
43. Sun, W., and Godson, G.N. (1998). Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis. Journal of molecular biology 276, 689-703.
44. Swart, J.R., and Griep, M.A. (1993). Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites. The Journal of biological chemistry 268, 12970-12976.
45. Salas, M., Mellado, R.P., and Vinuela, E. (1978). Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29. Journal of molecular biology 119, 269-291.
46. Thirlway, J., and Soultanas, P. (2006). In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues. Journal of bacteriology 188, 1534-1539.
47. Thirlway, J., Turner, I.J., Gibson, C.T., Gardiner, L., Brady, K., Allen, S., Roberts, C.J., and Soultanas, P. (2004). DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings. Nucleic acids research 32, 2977-2986.
48. Tougu, K., and Marians, K.J. (1996). The interaction between helicase and primase sets the replication fork clock. The Journal of biological chemistry 271, 21398-21405.
49. Tsai, H.H., Huang, C.H., Lin, A.M., and Chen, C.W. (2008). Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei. Nucleic acids research 36, e62.
50. Volff, J.N., and Altenbuchner, J. (1998). Genetic instability of the Streptomyces chromosome. Molecular microbiology 27, 239-246.
51. Volff, J.N., Viell, P., and Altenbuchner, J. (1997). Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans. Molecular & general genetics : MGG 253, 753-760.
52. van der Ende, A., Baker, T.A., Ogawa, T., and Kornberg, A. (1985). Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. Proceedings of the National Academy of Sciences of the United States of America 82, 3954-3958.
53. Wu, C.A., Zechner, E.L., Reems, J.A., McHenry, C.S., and Marians, K.J. (1992). Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis. The Journal of biological chemistry 267, 4074-4083.
54. Yang, C.C., Huang, C.H., Li, C.Y., Tsay, Y.G., Lee, S.C., and Chen, C.W. (2002). The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins. Molecular microbiology 43, 297-305.
55. Yang, C.C., Sun, W.C., Wang, W.Y., Huang, C.H., Lu, F.S., Tseng, S.M., and Chen, C.W. (2013). Mutational analysis of the terminal protein Tpg of Streptomyces chromosomes: identification of the deoxynucleotidylation site. PloS one 8, e56322.
56. Yoda, K., and Okazaki, T. (1991). Specificity of recognition sequence for Escherichia coli primase. Molecular & general genetics : MGG 227, 1-8.
57. Zechner, E.L., Wu, C.A., and Marians, K.J. (1992). Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. II. Frequency of primer synthesis and efficiency of primer utilization control Okazaki fragment size. The Journal of biological chemistry 267, 4045-4053.
論文全文使用權限:同意授權於2015-09-03起公開