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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李弘文 | |
dc.contributor.author | Cin-ya Chung | en |
dc.contributor.author | 鍾興雅 | zh_TW |
dc.date.accessioned | 2021-06-07T18:08:08Z | - |
dc.date.copyright | 2012-07-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-18 | |
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Allosteric effects of single-stranded and duplex DNA. J. Biol. Chem. 1992, 267, 7596-610. (8) Yarranton, G. T.; Gefter, M. L.: Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein. Proc. Natl. Acad. Sci. USA 1979, 76, 1658-62. (9) Truglio, J. J.; Karakas, E.; Rhau, B.; Wang, H.; DellaVecchia, M. J.; Van Houten, B.; Kisker, C.: Structural basis for DNA recognition and processing by UvrB. Nat. Struct. Mol. Biol. 2006, 13, 360-4. (10) Singleton, M. R.; Dillingham, M. S.; Gaudier, M.; Kowalczykowski, S. C.; Wigley, D. B.: Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 2004, 432, 187-93. (11) Spies, M.; Amitani, I.; Baskin, R. J.; Kowalczykowski, S. C.: RecBCD enzyme switches lead motor subunits in response to chi recognition. Cell 2007, 131, 694-705. (12) Boehmer, P. E.; Emmerson, P. T.: Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits. 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R.; Corzett, M.; Balhorn, R.; Yeh, Y.; Kowalczykowski, S. C.; Baskin, R. J.: Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 2001, 409, 374-8. (29) Finkelstein, I. J.; Visnapuu, M. L.; Greene, E. C.: Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 2010, 468, 983-7. (30) Dohoney, K. M.; Gelles, J.: Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 2001, 409, 370-4. (31) Perkins, T. T.; Li, H. W.: Single-molecule studies of RecBCD. Methods Mol. Biol. 2010, 587, 155-72. (32) Wu, C. G.; Bradford, C.; Lohman, T. M.: Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor. Nat. Struct. Mol. Biol. 2010, 17, 1210-7. (33) Bianco, P. R.; Kowalczykowski, S. C.: Translocation step size and mechanism of the RecBC DNA helicase. Nature 2000, 405, 368-72. (34) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G.: Persistence Length of Single-Stranded DNA. Macromolecules 1997, 30, 5763-65. (35) Murphy, M. C.; Rasnik, I.; Cheng, W.; Lohman, T. M.; Ha, T.: Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 2004, 86, 2530-7. (36) Rechendorff, K.; Witz, G.; Adamcik, J.; Dietler, G.: Persistence length and scaling properties of single-stranded DNA adsorbed on modified graphite. J. Chem. Phys. 2009, 131, 095103. (37) Hays, L. B.; Magar, M. E.; Zimai, B. H.: Persistence Length of DNA. Biopolymers 1969, 8, 531-536. (38) Wang, M. D.; Yin, H.; Landick, R.; Gelles, J.; Block, S. M.: Stretching DNA with optical tweezers. Biophys. J. 1997, 72, 1335-46. (39) Roman, L. J.; Kowalczykowski, S. C.: Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry 1989, 28, 2863-73. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16284 | - |
dc.description.abstract | DNA 解螺旋酶在細胞中負責將雙股DNA解開,產生單股DNA,在DNA的轉錄、轉譯、複製及重組反應中都是非常重要的角色。解螺旋酶的作用機制目前還沒有被完整地定義出來。結構生物學指出,解螺旋酶解開雙股的過程很有可能與此酵素在單股DNA上的移動有關。本研究以RecBCD做為解螺旋酶的模型,利用單分子的方法直接測試解螺旋酶是否以單股DNA移位酶的機制進行解旋。RecBCD主要的功能在於起始同源重組修復反應,並且辨識外來直鏈雙股DNA加以水解。當細胞發生雙股DNA斷裂(double strand breaks)時,RecBCD會自斷裂處開始解旋並水解此雙股DNA。RecBCD由三個次單元組成:RecB(3'->5'解螺旋酶)、RecC、及RecD(5'->3'解螺旋酶)。在此我們以單分子栓球實驗來觀察單一RecBCD酵素在單股DNA上移動的情形。首先我們製造了長大於200個鹼基無二級結構的單股DNA,並在其兩側加上雙股DNA延長。藉由計算接在RecBCD上小球的布朗運動大小,判斷蛋白質行經至DNA的位置。我們觀察到小球的布朗運動一開始從大的晃動,減小至停止晃動並離開,代表著RecBCD能夠從一開始的雙股移動至單股的區域,再繼續前進直到第二段雙股DNA的底端。由不同方向的DNA架設,我們證明RecBCD確實是5'->3'及3'->5'的單股DNA移位酶。此外,當遇到兩段不同方向的單股DNA時,我們也證明RecBCD的RecB和RecD次單元可互相切換已達到繼續移動的目的。 | zh_TW |
dc.description.abstract | The E. coli RecBCD helicase initiates the repair of DNA double strand break in the homologous recombination pathway. RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB is a 3' -> 5' helicase and RecD is a 5' -> 3' helicase. How RecBCD unwinds and translocates along duplex DNA is not clearly defined. Here we used a single-molecule tethered particle motion (TPM) experiment to visualize the RecBCD helicase translocation over long distance single-stranded (ss) DNA. We first prepared DNA substrates containing a > 200 nt long, unstructured ssDNA gap flanked by a double-stranded DNA end for RecBCD loading. In the TPM experiments, the bead-labeled, biotinylated RecBCD helicases are found to recognize and bind to the blunt, double-stranded DNA end, and successfully translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion amplitude. Successful observation of RecBCD translocation over long ssDNA gap in either 3' -> 5' or 5' -> 3' direction indicates that wild-type RecBCD functions as an ssDNA translocase. In addition, RecBCD is able to switch its motors between 3'-ssDNA and 5'-ssDNA. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T18:08:08Z (GMT). No. of bitstreams: 1 ntu-101-R99223120-1.pdf: 5311814 bytes, checksum: 00417367a3a48c00ea4496422fe57aa2 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | CHAPTER 1: Introduction 1
1.1 DNA Helicase and Translocase 1 1.2 Helicase Unwinding Mechanism 2 1.3 RecBCD 4 1.4 Homologous Recombination 6 1.4.1 Double Strand Break 6 1.4.2 Recombinational Repair 7 1.4.3 RecBCD Initiates Homologous Recombination 8 1.4.4 The Recognition of Chi Sequence 10 1.5 Tethered Particle Motion (TPM) 12 1.6 Single-Molecule Studies on RecBCD Helicases 13 CHAPTER 2: Materials and Methods 18 2.1 DNA Substrate Preparation 18 2.1.1 DNA Sequence without Secondary Structure 18 2.1.2 Single-Gapped DNA Substrate 20 2.1.3 Double-Gapped DNA Substrate 24 2.1.4 PCR Conditions 28 2.2 RecBCD Protein Purification 31 2.2.1 Minimal Plate 32 2.2.2 2YT Media Supplemented w/ Thymine and Biotin 33 2.3 Bead Preparation 33 2.3.1 Streptavidin Beads 33 2.3.2 RecBCD-Streptavidin Bead Coupling 35 2.4 Slide Preparation 38 2.5 Surface Preparation 38 2.6 Buffer Conditions 39 2.7 Experimental Procedure 39 2.8 Control Experiments 40 2.8.1 Dig-Bio Control Experiment 40 2.8.2 ssDNA Gap Control Experiment 41 2.9 Data Analysis 42 2.9.1 RecBCD Time Trace 43 2.9.2 ssDNA Gap Control Experiment 44 CHAPTER 3: Results and Discussion 46 3.1 Motivation 46 3.2 RecBCD Translocation along dsDNA 50 3.3 RecBCD Translocation along ssDNA 52 3.3.1 789nt (3'-->5' ssDNA) Gapped Substrate 52 3.3.2 860nt (5'-->3' ssDNA) Gapped Substrate 54 3.3.3 ssDNA Translocation Rate 54 3.4 RecBCD Translocation along Double-Gapped DNA 56 3.4.1 1655nt (5'-->3'/3'-->5') Double-Gapped DNA 57 3.4.2 1677nt (3'-->5'/5'-->3') Double-Gapped DNA 60 3.5 Discussion 62 CHAPTER 4: Conclusion 67 Appendix 68 References 84 | |
dc.language.iso | en | |
dc.title | 研究RecBCD解螺旋酶做為單股DNA移位酶之機制 | zh_TW |
dc.title | Investigating ssDNA Translocase Activities of RecBCD Helicase | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 謝道時,冀宏源,范秀芳 | |
dc.subject.keyword | RecBCD,DNA解螺旋酶,DNA移位酶,單分子栓球實驗, | zh_TW |
dc.subject.keyword | RecBCD,helicase,translocase,TPM, | en |
dc.relation.page | 86 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-07-18 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
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