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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 冀宏源(Hung-Yuan Chi) | |
dc.contributor.author | Yi-Zhen Jiang | en |
dc.contributor.author | 江宜蓁 | zh_TW |
dc.date.accessioned | 2021-06-08T02:46:07Z | - |
dc.date.copyright | 2018-01-04 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-10-14 | |
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Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background. Mol Cell Biol 19, 8686-8693. Sigurdsson, S., Van Komen, S., Bussen, W., Schild, D., Albala, J.S., and Sung, P. (2001). Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange. Genes Dev 15, 3308-3318. Slupianek, A., Jozwiakowski, S.K., Gurdek, E., and Skorski, T. (2009). BCR/ABL kinase interacts with and phosphorylates the RAD51 paralog, RAD51B. Leukemia 23, 2308-2310. Somyajit, K., Saxena, S., Babu, S., Mishra, A., and Nagaraju, G. (2015). Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart. Nucleic Acids Res 43, 9835-9855. Somyajit, K., Subramanya, S., and Nagaraju, G. (2010). RAD51C: a novel cancer susceptibility gene is linked to Fanconi anemia and breast cancer. Carcinogenesis 31, 2031-2038. Takata, M., Sasaki, M.S., Sonoda, E., Fukushima, T., Morrison, C., Albala, J.S., Swagemakers, S.M., Kanaar, R., Thompson, L.H., and Takeda, S. (2000). The Rad51 paralog Rad51B promotes homologous recombinational repair. Mol Cell Biol 20, 6476-6482. Takata, M., Sasaki, M.S., Tachiiri, S., Fukushima, T., Sonoda, E., Schild, D., Thompson, L.H., and Takeda, S. (2001). Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol 21, 2858-2866. Taylor, M.R., Spirek, M., Chaurasiya, K.R., Ward, J.D., Carzaniga, R., Yu, X., Egelman, E.H., Collinson, L.M., Rueda, D., Krejci, L., et al. (2015). Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination. Cell 162, 271-286. Taylor, M.R., Spirek, M., Jian Ma, C., Carzaniga, R., Takaki, T., Collinson, L.M., Greene, E.C., Krejci, L., and Boulton, S.J. (2016). A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling. Mol Cell 64, 926-939. Tebbs, R.S., Zhao, Y., Tucker, J.D., Scheerer, J.B., Siciliano, M.J., Hwang, M., Liu, N., Legerski, R.J., and Thompson, L.H. (1995). Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene. Proc Natl Acad Sci U S A 92, 6354-6358. Thacker, J. (2005). The RAD51 gene family, genetic instability and cancer. Cancer Lett 219, 125-135. Yokoyama, H., Sarai, N., Kagawa, W., Enomoto, R., Shibata, T., Kurumizaka, H., and Yokoyama, S. (2004). Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex. Nucleic Acids Res 32, 2556-2565. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20356 | - |
dc.description.abstract | 同源重組 (homologous recombination) 主要透過修復去氧核醣核酸雙股斷裂 (DNA double-strand breaks) 來維持基因體的穩定。在同源重組中,演化上被高度保留的 RAD51 重組酵素 (RAD51 recombinase) 是執行同源重組反應來修復雙股斷裂的去氧核醣核酸所不可或缺的。除此之外,研究發現在細胞內除了 RAD51 之外,去氧核醣核酸的修復還需要另外五個 RAD51 的同源蛋白質 (paralog) 形成兩個主要的蛋白質複合體,分別是 BCDX2 以及 CX3。目前已知這些 RAD51 的同源蛋白質與 RAD51 約有二十至三十百分比的胺基酸序列相似性,並且也具有結合與水解三磷酸腺苷 (ATP) 的能力。然而,由於過去純化 BCDX2 蛋白質複合體的困難,導致目前關於 BCDX2 複合體的生化特性以及與 RAD51 作用相關的詳細機制仍有許多未知。在我們的研究中,我們成功地建立了表達和純化 BCDX2 複合體重組蛋白質的系統,並且得到了 BCDX2 複合體重組蛋白質。我們的研究證明了重組蛋白質 BCDX2 複合體具有結合單股去氧核醣核酸 (single-strand DNA) 的能力也和 RAD51 有直接的蛋白質交互作用。 然而,出乎我們意料之外,純化的 BCDX2 蛋白質複合體,特別是 RAD51B 能夠在體外實驗被磷酸化 (phosphorylation),並且我們初步的實驗證明 RAD51B 的磷酸化能夠影響 BCDX2 複合體與 RAD51 的交互作用。 | zh_TW |
dc.description.abstract | Homologous recombination (HR) plays a central role in maintaining genomic integrity by repairing DNA double-strand breaks (DSBs). The evolutionarily conserved RAD51 recombinase is the key enzyme to execute the recombination-mediated DSB repair. Moreover, cell-based studies have well documented that five RAD51 paralogs forming two distinct complexes BCDX2 and CX3 are required for RAD51 activity. These RAD51 paralogs share about 20-30% sequence identity at amino acid level with RAD51 and possess the ability of ATP binding and hydrolysis. However, it remains largely unknown about the mechanistic perspective of the BCDX2 complex in regulating RAD51 activity due to the difficulty of obtaining BCDX2 protein complex. Here we successfully established expression and purification procedures of the BCDX2 complex in the human expression system. The purified BCDX2 complex exhibits the single-strand DNA binding activity and physically interacts with RAD51. To our surprise, we found that RAD51B could be phosphorylated in vitro. Our preliminary data indicates that the phosphorylation of RAD51B affects the protein-protein interaction between BCDX2 complex and RAD51 recombinase. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:46:07Z (GMT). No. of bitstreams: 1 ntu-106-R04b46004-1.pdf: 5168258 bytes, checksum: ce43fc406887ecf0c296ebb81915f2f8 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 中文摘要 1
ABSTRACT 3 CHAPTER 1: INTRODUCTION 9 1-1 The mechanisms of homologous recombination (HR) 9 1-2 Identification of RAD51 paralogs, BCDX2 and CX3 complex as HR factors 10 1-3 The functional roles of BCDX2 complex in HR 12 1-4 Motivation of my thesis studies 13 CHAPTER 2: MATERIALS AND METHODS 15 2-1 Oligonucleotides 15 2-2 Plasmids 15 2-2-1 Generation of pcDNA3.4-tetrad vector 15 2-2-2 Construct RAD51BCDX2, RAD51B-C, and RAD51D-X2 expression plasmids 15 2-3 Protein expression and purification 16 2-3-1 BCDX2 protein complex 16 2-3-2 BC sub-complex 18 2-3-3 DX2 sub-complex 19 2-4 Electrophoretic mobility shift assay 20 2-5 ATP binding assay 20 2-6 Kinase assay 21 2-7 Affinity pull down assay 21 CHAPTER 3: RESULTS 23 3-1 Purification of BCDX2 complex in Expi293TM human cell line 23 3-2 Purified BCDX2 complex has single-strand DNA binding activity 24 3-3 ATP binding assay of BCDX2 complex 25 3-4 BCDX2 complex is phosphorylated in vitro by unknown kinases present in the purified protein mixture 26 3-5 Specific kinases were co-pulled down during BCDX2 purification 27 3-6 The possible phosphorylation sites of RAD51B 28 3-7 Purified BCDX2 complex has species-specific interaction with RAD51 29 3-8 In vitro phosphorylation abolishes the interaction between BCDX2 and RAD51 30 CHAPTER 4: CONCLUSIONS AND DISCUSSIONS 31 4-1 Summary of key findings 31 4-2 Discussion and future directions 31 4-2-1 Examine the impact of RAD51B phosphorylation on homologous recombination 31 4-2-2 Investigate the regulation of BCDX2 complex by specific kinases 33 4-2-3 Functional role of BCDX2 complex in RAD51-mediated recombination 34 FIGURES 36 TABLES 45 REFERENCES 47 APPENDIX 52 | |
dc.language.iso | en | |
dc.title | 探討 BCDX2 蛋白質複合體的生化特性與其與 RAD51 重組酵素的交互作用 | zh_TW |
dc.title | Investigate the Biochemical Characteristic of BCDX2 Complex and Its Functional Role with RAD51 | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李弘文(Hung-Wen Li),游佳融 | |
dc.subject.keyword | 去氧核醣核酸雙股斷裂,去氧核醣核酸修復,同源重組,重組酵素 RAD51,RAD51B-RAD51C-RAD51D-XRCC2 蛋白質複合體,BCDX2 蛋白質複合體,磷酸化, | zh_TW |
dc.subject.keyword | DNA double-strand breaks,DNA repair,Homologous recombination,RAD51,RAD51B-RAD51C-RAD51D-XRCC2 complex,BCDX2 complex,Phosphorylation, | en |
dc.relation.page | 56 | |
dc.identifier.doi | 10.6342/NTU201704280 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-10-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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