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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
dc.contributor.author | Sheng-Yao Lin | en |
dc.contributor.author | 林聖堯 | zh_TW |
dc.date.accessioned | 2021-07-11T14:37:45Z | - |
dc.date.available | 2019-08-30 | |
dc.date.copyright | 2017-08-30 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-07 | |
dc.identifier.citation | 1. Sonoda, E.; Sasaki, M. S.; Buerstedde, J. M.; Bezzubova, O.; Shinohara, A.; Ogawa, H.; Takata, M.; Yamaguchi-Iwai, Y.; Takeda, S., Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 1998, 17 (2), 598-608.
2. Hsu, H. F.; Ngo, K. V.; Chitteni-Pattu, S.; Cox, M. M.; Li, H. W., Investigating Deinococcus radiodurans RecA protein filament formation on dsDNA by a real-time single-molecule approach. Biochemistry 2011, 50 (39), 8270-80. 3. Sung, P.; Klein, H., Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 2006, 7 (10), 739-50. 4. Qi, Z.; Redding, S.; Lee, J. Y.; Gibb, B.; Kwon, Y. H.; Niu, H.; Gaines, W. A.; Sung, P.; Greene, E. C., DNA sequence alignment by microhomology sampling during homologous recombination. Cell 2015, 160 (5), 856-69. 5. Masson, J. Y.; West, S. C., The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem Sci 2001, 26 (2), 131-6. 6. Borgogno, M. V.; Monti, M. R.; Zhao, W.; Sung, P.; Argarana, C. E.; Pezza, R. J., Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange. J Biol Chem 2016, 291 (10), 4928-38. 7. Lee, J. Y.; Terakawa, T.; Qi, Z.; Steinfeld, J. B.; Redding, S.; Kwon, Y. H.; Gaines, W. A.; Zhao, W.; Sung, P.; Greene, E. C., Base triplet stepping by the Rad51/RecA family of recombinases. Science 2015, 349 (6251), 977-81. 8. Masson, J.-Y.; Davies, A. A.; Hajibagheri, N.; Van Dyck, E.; Benson, F. E.; Stasiak, A. Z.; Stasiak, A.; West, S. C., The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J 1999, 18 (22), 6552-6560. 9. Sehorn, M. G.; Sigurdsson, S.; Bussen, W.; Unger, V. M.; Sung, P., Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature 2004, 429 (6990), 433-437. 10. Brown, M. S.; Grubb, J.; Zhang, A.; Rust, M. J.; Bishop, D. K., small Rad51 and Dmc1 complexes often Co-occupy both ends of a meiotic DNA double strand break. PLOS Genetics 2016, 11 (12), e1005653. 11. Sauvageau, S.; Stasiak, A. Z.; Banville, I.; Ploquin, M.; Stasiak, A.; Masson, J.-Y., fission yeast Rad51 and Dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments. Mol. Cell. Biol. 2005, 25 (11), 4377-4387. 12. Machaca, K., Ca(2+) signaling, genes and the cell cycle. Cell Calcium 2010, 48 (5), 243-50. 13. Bugreev, D. V.; Mazin, A. V., Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (27), 9988-93. 14. Bugreev, D. V.; Golub, E. I.; Stasiak, A. Z.; Stasiak, A.; Mazin, A. V., Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J Biol Chem 2005, 280 (29), 26886-95. 15. Lee, M. H.; Chang, Y. C.; Hong, E. L.; Grubb, J.; Chang, C. S.; Bishop, D. K.; Wang, T. F., Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA. J Biol Chem 2005, 280 (49), 40980-4. 16. Chung, C.; Li, H.-W., Direct observation of RecBCD helicase as single-stranded DNA translocases. J. Am. Chem. Soc. 2013, 135 (24), 8920-8925. 17. Fan, H. F.; Li, H. W., Studying RecBCD helicase translocation along Chi-DNA using tethered particle motion with a stretching force. Biophys J 2009, 96 (5), 1875-83. 18. Fan, H. F.; Cox, M. M.; Li, H. W., Developing single-molecule TPM experiments for direct observation of successful RecA-mediated strand exchange reaction. PLOS ONE 2011, 6 (7), e21359. 19. Fan, H. F.; Cheng, Y. S.; Ma, C. H.; Jayaram, M., Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination. Nucleic Acids Res. 2015, 43 (6), 3237-55. 20. Piechura, J. R.; Tseng, T. L.; Hsu, H. F.; Byrne, R. T.; Windgassen, T. A.; Chitteni-Pattu, S.; Battista, J. R.; Li, H. W.; Cox, M. M., Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation. DNA Repair (Amst) 2015, 26, 30-43. 21. Yu, X.; Egelman, E. H., Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale. J Mol Biol 2010, 401 (3), 544-51. 22. Hilario, J.; Amitani, I.; Baskin, R. J.; Kowalczykowski, S. C., Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (2), 361-368. 23. Busygina, V.; Gaines, W. A.; Xu, Y.; Kwon, Y.; Williams, G. J.; Lin, S. W.; Chang, H. Y.; Chi, P.; Wang, H. W.; Sung, P., Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1. DNA Repair (Amst) 2013, 12 (9), 707-12. 24. Chang, H. Y.; Liao, C. Y.; Su, G. C.; Lin, S. W.; Wang, H. W.; Chi, P., Functional relationship of ATP hydrolysis, presynaptic filament stability, and homologous DNA pairing activity of the human meiotic recombinase DMC1. J Biol Chem 2015, 290 (32), 19863-73. 25. Bishop, D. K., RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell 1994, 79 (6), 1081-92. 26. Gasior, S. L.; Wong, A. K.; Kora, Y.; Shinohara, A.; Bishop, D. K., Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes Dev 1998, 12 (14), 2208-21. 27. Busygina, V.; Sehorn, M. G.; Shi, I. Y.; Tsubouchi, H.; Roeder, G. S.; Sung, P., Hed1 regulates Rad51-mediated recombination via a novel mechanism. Genes Dev 2008, 22 (6), 786-95. 28. Cloud, V.; Chan, Y. L.; Grubb, J.; Budke, B.; Bishop, D. K., Dmc1 catalyzes interhomolog joint molecule formation in meiosis with Rad51 and Mei5-Sae3 as accessory factors. Science 2012, 337 (6099), 1222-5. 29. Neuman, K. C.; Nagy, A., Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 2008, 5 (6), 491-505. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77934 | - |
dc.description.abstract | 雙股斷裂的DNA損壞,可利用同源重組(homologous recombination)的方式,準確地修復受損的DNA。DNA重組酶催化受損DNA與同源的雙股DNA配對,進行股交換反應,進而利用完整的DNA模板進行修復。真核生物具有兩種同源重組酶Dmc1 與Rad51,其序列及功能大多相似,但兩者在細胞週期出現的時間點卻有不同。在細胞分裂時(mitosis)只需Rad51即可完成DNA修復,然而在減數分裂(meiosis)的過程中,則同時需要Rad51與Dmc1兩者的參與。為何不同的細胞階段對DNA重組酶的需求不同,其機制仍然未明。利用單分子栓球實驗(single molecule tethered particle motion),本研究直接比較Saccharomyces cerevisiae 酵母菌中Rad51與Dmc1兩個重組酶在形成核蛋白絲階段的動力學。我們觀察到ScRad51與ScDmc1在核蛋白絲的形成動力學明顯不同。在核蛋白絲的形成過程,成核的階段是速率決定步驟,而ScRad51的成核速率比ScDmc1來的快,但兩者在其核蛋白絲的延展及核蛋白絲的長度上,則沒有差別。不同的重組酶濃度實驗中指出,ScRad51與ScDmc1皆以二倍體(dimer)的形式進行成核,因此成核速率的差異是來自於ScRad51對單股DNA (ssDNA)有較高的親和力,這個現象也反應在ScRad51的成核速率隨著不同長度的單股DNA而明顯遞增。相對來說,ScDmc1的成核速率在不同長度的單股DNA中則沒有明顯關聯,而是與單雙股交界處的連接點(ds/ss DNA junction)處的成核速率相似。Rad51與Dmc1核蛋白絲的形成動力學上的差異,及其對於不同DNA構型的偏好性,在建構同源重組在減數分裂過程中進行的分子模型時,可以提供這兩種DNA重組酶角色的重要基礎。 | zh_TW |
dc.description.abstract | The double-stranded break (DSB) DNA damage can be repaired in high fidelity using homologous recombination (HR) pathway. DNA recombinases form nucleoprotein filaments and catalyze the pairing of the homologous DNA sequence and the exchange of DNA strands. So this allow DNA replication to repair the damaged DNA using the homologous DNA template. In eukaryotes, there exist two recombinases, Rad51 and Dmc1. Both of them share similar amino acid sequences and functions. However, while only Rad51 is required in mitosis, both Rad51 and Dmc1 are essential in meiosis recombination. The mechanism underlying the differential requirement is unknown. Here, we compared the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae ScRad51 and ScDmc1 using single-molecule tethered particle motion experiments (TPM). We found an apparent kinetics difference during nucleoprotein filament assembly for ScRad51 and ScDmc1. Forming recombinase nuclei in single-stranded (ss) DNA is the rate-limited step in the nucleoprotein filament assembly. In our real-time assembly measurement, we found that ScRad51 has much faster nucleation rate than ScDmc1, while the extension time and filament coverage are similar for both ScRad51 and ScDmc1. Study of the nucleation times at different recombinase concentrations showed that ScRad51 and ScDmc1 have the similar power-law dependence of ~ 2, suggesting that both form stable nuclei in dimers. Therefore, the faster nucleation of ScRad51 likely results from the higher ssDNA affinity. This is consistent with the observation that nucleation times of ScRad51 increases with DNA substrates of longer ssDNA lengths. However, nucleation times of ScDmc1 did not show apparent ssDNA length dependence. These kinetic differences in nucleoprotein filament assembly provide important molecular constrains in explaining biochemical roles of Rad51 and Dmc1. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:37:45Z (GMT). No. of bitstreams: 1 ntu-106-R04223130-1.pdf: 2792178 bytes, checksum: bbea9b6cbe519b4434ce34dc9864cce6 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 摘要 i
ABSTRACT ii TABLE OF CONTENTS iii LIST OF FIGURES v LIST OF TABLES vi CHAPTER 1. INTRODUCTION 1 1-1 Homologous recombination (HR) 1 1-2 Dmc1 and Rad51 Recombinase 3 1-3 Calcium modulates the activity of Rad51 and Dmc1 5 1-4 Motivation of this study 7 CHAPTER 2. MATERIALS AND METHODS 8 2-1 Rad51, Dmc1, Mei5-Sae3 protein purification 8 2-2 gap DNA dT200, AC263 synthesis method 8 2-2.1 AC263 gap DNA synthesis 8 2-2.2 dT gap synthesis 8 2-3 Buffer Recipe 10 2-4 Single-molecule Tethered Particle Motion (TPM) 11 2-4.1 microscope parameters 12 2-4.2 Reaction slide preparation 13 2-5 Data Analysis 13 2-5.1 Brownian Motion (BM) amplitude analysis 13 2-5.2 Nucleation time-trace analysis 14 2-5.3 Nucleation time determination 15 2-5.4 TPM snapshots 15 CHAPTER 3. RESULT 17 3-1.ScRad51 has fast nucleation rate than Dmc1 17 3-2 dsDNA assembly control comparison between Rad51 and Dmc1 19 3-3 Dmc1-DNA interaction is different from Rad51-DNA. 21 3-4 The nucleation rates of ScDmc1 and ScRad51 have the similar recombinase-concentration dependence. 22 3-5 Rad51 has higher assembly preference on ssDNA than Dmc1 25 3-6 Calcium stimulates Dmc1’s nucleation 29 3-7 ScRad51 doesn’t stimulate the nucleation step of ScDmc1. 31 CHAPTER 4.CONCLUSION AND OUTLOOK 34 4-1 Conclusion 34 4-2 Outlook 37 Reference 39 Appendix 43 | |
dc.language.iso | en | |
dc.title | 利用單分子技術探討DNA重組酶Rad51與Dmc1核蛋白絲之成核動力學 | zh_TW |
dc.title | Studying the Assembly Kinetics of Saccharomyces cerevisiae Rad51 and Dmc1 Recombinases Using Single Molecule Methods | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 范秀芳(Hsiu-Fang Fan),冀宏源(Hung-Yuan Chi),林敬哲(Jing-Jer Lin),王廷方(Ting-Fang Wang),陳昭岑(Chao-Tsen Chen) | |
dc.subject.keyword | DNA重組?,Rad51,Dmc1,核蛋白絲,DNA同源重組修護,單分子栓球實驗, | zh_TW |
dc.subject.keyword | DNA Recombinases,Rad51,Dmc1,nucleoprotein filament,DNA homologous recombinational repair,single molecule Tethered Particle Motion, | en |
dc.relation.page | 43 | |
dc.identifier.doi | 10.6342/NTU201702627 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-07 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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