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研究生:林柏廷
研究生(外文):Bo-Ting Lin
論文名稱:探討Nucleostemin生化特性及與重組酵素RAD51的交互作用
論文名稱(外文):Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair
指導教授:冀宏源
口試委員:李弘文鄭淑珍譚婉玉廖泓鈞
口試日期:2017-05-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:46
中文關鍵詞:Nucleostemin雙股去氧核醣核酸斷裂同源重組RAD51重組酵素三磷酸鳥苷結合蛋白
外文關鍵詞:NucleosteminDouble-strand breaksHomologous recombinationRAD51 recombinaseGTP binding protein
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染色體複製(replication)是細胞增生的重要步驟,在具有高度增生能力的細胞中,頻繁的複製過程容易造成因複製叉停滯(stalled replication fork),或是複製叉崩塌(collapse of replication fork)而形成的雙股去氧核醣核酸斷裂斷裂(DNA double-strand breaks),進而導致基因的不穩定。在過去研究發現,Nucleostemin (NS)參與在複製所產生雙股去氧核醣核酸斷裂的修復機制中。NS是屬於三磷酸鳥苷結合蛋白並在高度增生細胞中大量表達,目前已知若細胞中缺乏NS,複製過程將累積大量自發性的雙股去氧核醣核酸斷裂;另一方面,過度表達NS則能夠減少在複製機制中hydroxyurea所引發的雙股去氧核醣核酸斷裂。值得注意的是在近期的研究報導,NS與修復雙股去氧核醣核酸斷裂的重組酵素RAD51同時存在於相同的免疫沉澱體;且NS的缺少更進一步地影響RAD51在斷裂染色體上存在的數量,顯示NS參與在RAD51相關的修復機制之中。然而過去由於純化NS蛋白相當困難,使其本身蛋白生化特性以及與RAD51之間交互作用以完成修復的詳細機制仍有許多未知。在我們的研究中,我們建立了表達與純化NS重組蛋白的系統,並且成功地得到了高純度的NS蛋白質,利用NS重組蛋白,我們證明了NS能夠與三磷酸鳥苷結合。在凝膠遷移實驗中發現,NS具有去氧核醣核酸的結合能力並且傾向與雙股去氧核醣核酸結合。根據親和性沉降測試,我們發現哺乳類的NS與RAD51有直接的蛋白質交互作用,但不會與原核生物的重組酵素RecA進行交互作用。然而NS的去氧核醣核酸結合能力以及與RAD51交互作用對於NS所參與的去氧核醣核酸修復機制為何,仍然需要進一步分析。
Cells constantly suffer various types of DNA damages including DNA double-strand breaks (DSBs). Notably, spontaneous DSBs are frequently generated in highly proliferating cells when replication forks were stalled or collapsed. Recent cell-based and animal studies have documented that nucleostemin (NS) participates in the DSB repair including replication-induced DSBs. NS belongs a G protein family and the expression is highly enriched in proliferating cells. Depletion of NS accumulates the spontaneous DSBs in S phase cells; conversely, overexpression of NS can significantly reduce the DSBs generated by stalled replication-fork upon the treatment of hydroxyurea. Moreover, NS forms a DSB-induced focus and interacts with RAD51, the key enzyme of homologous recombination-mediated DSB repair pathway. Interestingly, depletion of NS will significantly attenuate the recruitment of RAD51 to DSBs. Taken all results together, it is clear that nucleostemin participates in the RAD51-mediated recombination repair. However, it remains largely unknown regarding the NS biochemical characteristics and its functional interaction with RAD51-mediated DNA exchange, due to the hurdle of obtaining the NS recombinant proteins for the biochemical and functional analyses. Here, we have successfully established the expression and purification procedures of NS. Our biochemical analyses demonstrated that the purified monodispersed NS protein possesses a GTP binding ability. To our surprise, nucleostemin binds DNA with a preference for duplex DNA rather than single-strand DNA. Importantly, a direct protein-protein interaction between purified NS and RAD51 recombinase has been observed. Notably, we showed that the physical interaction is a species-specific since no interaction has been detected between mammalian NS and prokaryotic RecA recombinase. The functional significances of DNA binding and RAD51 interaction by NS on RAD51-mediated DSB repair will be examined in the near future. In summary, our NS purification system and the biochemical properties reported herein should expedite further mechanistic study regarding the mechanistic action of NS in RAD51-mediated DSB repair.
論文口試委員審定書....................................................I
誌謝.................................................................II
中文摘要............................................................III
ABSTRACT.........................................................IV

CHAPTER 1: INTRODUCTION.........................................1
1-1 Proliferating cell enriched protein: Nucleostemin (NS) .....................1
1-2 Molecular property of NS: a GTP-binding protein .........................1
1-3 Biological significance of nucleostemin .................................3
1-4 Homologous recombination and RAD51 recombinase .....................5
1-5 Motivation of my study .............................................7

CHAPTER 2: MATERIALS AND METHODS ..............................8
2-1 Plasmids .........................................................8
2-1.1 NS expression plasmids .........................................8
2-1.2 RAD51 expression plasmids .....................................8
2-1.3 Truncated NS variants expression plasmids ..........................8
2-2 Protein expression and purification ....................................9
2-2.1 Nucleostemin recombinant protein .................................9
2-2.2 RAD51 recombinant protein .....................................10
2-2.3 Expression and purification of truncated nucleostemin variants ..........11
2-3 DNA substrates ..................................................12
2-3.1 32P-labeled 40mer ssDNA .......................................12
2-3.2 32P-labeled 40mer dsDNA .......................................13
2-4 GTP-Agarose pull-down assay .......................................13
2-5 DNA mobility shift assay ...........................................14
2-6 Affinity pull down assay ............................................14

CHAPTER 3: RESULTS ...............................................16
3-1 Expression and purification of nucleostemin ............................16
3-2 GTP binding activity of nucleostemin .................................17
3-3 DNA binding activity of nucleostemin .................................17
3-4 Nucleostemin harbors multiple DNA binding sites .......................19
3-5 Nucleostemin physically interacts with recombinase RAD51 ...............20
3-6 Nucleostemin interacts with RAD51 via multiple contact sites ..............21
3-7 GTP alter the interaction between nucleostemin and RAD51 ................22

CHAPTER 4: CONCLUSION AND DISCUSSION .........................23
4-1 Summary of key findings ...........................................23
4-2 Discussion and Future Direction .....................................23
4-2.1 Oligomeric status of nucleostemin ................................23
4-2.2 GTP binding and hydrolysis of nucleostemin ........................24
4-2.3 The nucleic acids binding property: DNA and RNA ...................25
4-2.4 Functional interaction of nucleostemin and RAD51 ...................28

FIGURE LEGENDS ..................................................30
REFERENCE ........................................................37
APPENDIX ..........................................................41
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