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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 冀宏源 | zh_TW |
| dc.contributor.advisor | Hung-Yuan Chi | en |
| dc.contributor.author | 高誌遠 | zh_TW |
| dc.contributor.author | Chih-Yuan Kao | en |
| dc.date.accessioned | 2024-03-07T16:26:17Z | - |
| dc.date.available | 2024-03-08 | - |
| dc.date.copyright | 2024-03-07 | - |
| dc.date.issued | 2016 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1. Benson, F.E., Stasiak, A., and West, S.C. (1994). Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J 13, 5764-5771.
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New, J.H., Sugiyama, T., Zaitseva, E., and Kowalczykowski, S.C. (1998). Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391, 407-410. 26. Passy, S.I., Yu, X., Li, Z.F., Radding, C.M., Masson, J.Y., West, S.C., and Egelman, E.H. (1999). Human Dmc1 protein binds DNA as an octameric ring. P Natl Acad Sci USA 96, 10684-10688. 27. Pezza, R.J., Voloshin, O.N., Vanevski, F., and Camerini-Otero, R.D. (2007). Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing. Genes Dev 21, 1758-1766. 28. Raschle, M., Van Komen, S., Chi, P., Ellenberger, T., and Sung, P. (2004). Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54. J Biol Chem 279, 51973-51980. 29. Say, A.F., Ledford, L.L., Sharma, D., Singh, A.K., Leung, W.K., Sehorn, H.A., Tsubouchi, H., Sung, P., and Sehorn, M.G. (2011). The budding yeast Mei5-Sae3 complex interacts with Rad51 and preferentially binds a DNA fork structure. DNA Repair (Amst) 10, 586-594. 30. Schwacha, A., and Kleckner, N. (1997). Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway. Cell 90, 1123-1135. 31. Sehorn, M., and Sehorn, H. (2011). Visualization of Human Dmc1 Presynaptic Filaments. Methods Mol Biol 745, 485-496. 32. Seong, C., Colavito, S., Kwon, Y., Sung, P., and Krejci, L. (2009). Regulation of Rad51 recombinase presynaptic filament assembly via interactions with the Rad52 mediator and the Srs2 anti-recombinase. J Biol Chem 284, 24363-24371. 33. Shinohara, A., Gasior, S., Ogawa, T., Kleckner, N., and Bishop, D.K. (1997). Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination. Genes Cells 2, 615-629. 34. Shinohara, A., Ogawa, H., and Ogawa, T. (1992). Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457-470. 35. Shinohara, A., and Ogawa, T. (1998). Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391, 404-407. 36. Shinohara, A., and Shinohara, M. (2004). Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination. Cytogenet Genome Res 107, 201-207. 37. Shinohara, M., Gasior, S.L., Bishop, D.K., and Shinohara, A. (2000). Tid1/Rdh54 promotes colocalization of rad51 and dmc1 during meiotic recombination. Proc Natl Acad Sci U S A 97, 10814-10819. 38. Sung, P. (1994). Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241-1243. 39. Sung, P., and Klein, H. (2006). Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739-750. 40. Tarsounas, M., Morita, T., Pearlman, R.E., and Moens, P.B. (1999). RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes. J Cell Biol 147, 207-220. 41. Tsubouchi, H., and Roeder, G.S. (2004). The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination. Genetics 168, 1219-1230. 42. Tsubouchi, H., and Roeder, G.S. (2006). Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired. Genes Dev 20, 1766-1775. 43. Yu, D.S., Sonoda, E., Takeda, S., Huang, C.L., Pellegrini, L., Blundell, T.L., and Venkitaraman, A.R. (2003). Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2. Mol Cell 12, 1029-1041. 44. Zhao, W., and Sung, P. (2015). Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis. Nucleic Acids Res 43, 4055-4066. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92176 | - |
| dc.description.abstract | 同源重組(homologous recombination)是細胞修復雙股去氧核醣核酸斷裂(DNA double-strand breaks)的重要機制，用以確保基因組的正確性及完整。細胞亦會透過減數分裂時的重組反應(meiotic recombination)使同源染色體能正確的配對與分離；並且產生子代的基因多樣性。為了執行減數分裂時的重組反應，幾乎所有的真核生物都會在減數分裂時表現兩種重組酵素(recombinase): Rad51和Dmc1。過去研究已知若失去Dmc1的功能，將無法正確執行減數分裂時的重組反應。此外，最近的研究則指出，Rad51可能扮演輔助因子(accessory factor)的角色，協助Dmc1有效進行減數分裂時的重組反應。然而Rad51與Dmc1是如何相互作用並完成減數分裂時的重組反應，其詳細機制仍是未知。為了研究這個問題，我們希望可以從兩個面向來探討，其一是了解這兩種重組酵素是如何進行交互作用，其二是探究此交互作用的功能性為何。我們的研究結果發現，在水溶液中Dmc1 與Rad51有直接的蛋白和蛋白的交互作用，但不會與原核生物的重組酵素RecA發生交互作用。我們也利用點突變法以及蛋白質截斷試驗兩種實驗方法，觀察到Rad51與Dmc1之間具有多個交互作用的位點，並且推測可能不是透過 ”球窩關節模型” (ball-and-socket model)的方式產生此彼此的交互作用。根據核酸內切酶保護實驗，我們發現Rad51具有較Dmc1更佳的單股DNA親和能力，因此我們假設Rad51透過與Dmc1發生交互作用，可以幫助Dmc1與DNA結合。然而我們並沒有觀察到在Rad51與Dmc1發生交互作用後，對DNA免於核酸內切酶剪切的相乘(synergistic effect)保護效果。綜合上述我們的研究，顯示Rad51與Dmc1在與DNA結合之前，可能會發生交互作用；我們的研究也提供了未來更多的研究方向，去探討Rad51與Dmc1可能的功能性交互作用。 | zh_TW |
| dc.description.abstract | Meiotic recombination plays essential role in eukaryotes for generating genetic diversity and proper chromosome segregation. For conducting meiotic recombination, most eukaryotes have two recombinases, Rad51 and Dmc1. It has been well documented that meiotic recombination fails in Dmc1-deficient yeast. Moreover, a recent study suggested that Rad51 is an accessory factor of Dmc1 for efficient meiotic recombination. However, the detailed mechanism of how these two proteins cooperating in meiotic recombination remains unclear. To address this question, we wish to (1) determine the interaction mode of Rad51 and Dmc1; and (2) decipher whether the functional interaction is within two recombinases. We made a significant contribution in demonstrating that Dmc1 interacts with Rad51 directly in solution and has no interaction with prokaryotic recombinase, RecA. This result indicates that the physical interaction is conserved in eukaryotes. In addition, combining site-directed mutagenesis and protein truncation approaches, we observed that there are multiple interaction contacts within interaction surface of Rad51 and Dmc1 and hypothesized the interaction might be not through “ball-and-socket” mode. Moreover, in endonuclease protection assay, we demonstrate the higher DNA binding affinity of Rad51 than that of Dmc1 which possibly implies a hypothesis that Rad51 interacts with Dmc1 to help its filament assembly. However, no significant enhancement of Dmc1 filament assembly has been seen in the presence of Rad51. Taken together, our results imply that Rad51 interacts with Dmc1 before binding DNA and provide more directions for functional assays in future. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-03-07T16:26:17Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-03-07T16:26:17Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | TABLE OF CONTENTS
論文口試委員審定書 I 誌謝 II 中文摘要 III ABSTRACT V CHAPTER 1: INTRODUCTION 1 1-1 Biological Functions of Homologous Recombination (HR) 1 1-2 The Mechanism of HR in Meiosis 1 1-3 Two Eukaryotic Recombinases: Rad51 and Dmc1 2 1-4 The Functional Role of Rad51 and Dmc1 in Meiosis 4 1.5 Motivation of my study 6 CHAPTER 2: MATERIALS AND METHODS 7 2-1 Plasmids 7 2-1.1 Rad51 and Dmc1 expression plasmids 7 2-1.2 pGEX-6P-1-Rad51 truncated variants 7 2-1.3 Mei5-Sae3 expression plasmids 7 2-2 Protein expression and purification 7 2-2.1 Dmc1 7 2-2.2 Rad51 9 2-2.3 Rad51 truncated variants 9 2-2.4 Mei5-Sae3 11 2-3 DNA substrates 12 2-3.1 32P -labeled 80mer ssDNA 12 2-3.2 32P -labeled 331mer ssDNA 12 2-4 Endonuclease protection assay 14 2-5 Affinity pull-down assays 15 CHAPTER 3: RESULTS 17 3-1 Purification of Rad51 and Dmc1 17 3-2 Rad51 directly interacts with Dmc1 in solution 17 3-3 Rad51 interacts with Dmc1 via multiple contact points 19 3-4 Nucleoprotein filament of Rad51 is much stable than Dmc1 21 3-5 Rad51 has no promotion on the stability of Dmc1 nucleoprotein filament in endonuclease protection assay 22 CHAPTER 4: CONCLUSION AND DISCUSSION 24 4-1 Summary of key findings 24 4-2 Discussion 25 4-2.1 Interaction between Rad51 and Dmc1 might be with “ring-to-ring” mode 25 4-2.2 Functional interactions of Rad51 and Dmc1 28 4-2.3 Rad51 might change the DNA-binding preference of Dmc1 from double-stranded to single-stranded DNA 30 FIGURE LEGENDS 32 REFERENCE 40 APPENDIX 44 | - |
| dc.language.iso | en | - |
| dc.title | 探討重組酵素 Rad51 與Dmc1 的功能性交互作用 | zh_TW |
| dc.title | Investigating the Functional Interaction of Rad51 and Dmc1 Recombinases | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 104-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 李宏文;王廷方;王中茹 | zh_TW |
| dc.contributor.oralexamcommittee | Hung-Weng Li;Ting-Fang Wang;Chung-Ju Wang | en |
| dc.subject.keyword | 同源重組,減數分裂時的重組反應,Rad51重組酵素,Dmc1重組酵素,輔助因子,相乘作用, | zh_TW |
| dc.subject.keyword | Homologous recombination,meiotic recombination,Rad51,Dmc1,accessory factor,synergistic effect, | en |
| dc.relation.page | 50 | - |
| dc.identifier.doi | 10.6342/NTU201603365 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2016-08-21 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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