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標題: | 探討SWI5-SFR1 複合體在RAD51 參與的去氧核醣核酸修復的生化機制 The mechanistic study of SWI5-SFR1 complex on RAD51-mediated DNA repair |
作者: | Guan-Chin Su 蘇綸勤 |
指導教授: | 冀宏源(Hung-Yuan Chi) |
關鍵字: | 同源重組,DNA 雙股斷裂,RAD51 核蛋白絲,SWI5-SFR1,ATP 水解,ADP 釋放, Homologous recombination,DNA double-strand breaks,RAD51 presynaptic filament,SWI5-SFR1,ATP hydrolysis,ADP release, |
出版年 : | 2016 |
學位: | 博士 |
摘要: | 同源重組酵素RAD51 會進行同源重組去修復DNA 雙股斷裂。RAD51 會形成核蛋白絲來去啟動同源重組反應。最近哺乳類細胞實驗指出SWI5 和 SFR1 會形成複合體並參與在RAD51 所主導的同源重組修復。為了探討哺乳類的SWI5-SFR1 在RAD51 所主導的同源重組修復的生化角色,我們建立了蛋白表現及純化的步驟得到老鼠SWI5-SFR1 蛋白複合體。經由我們的生化研究指出,SWI5-SFR1 會刺激RAD51 所進行的同源DNA 配對以及穩定RAD51 核蛋白絲。這樣的研究顯示出SWI5-SFR1 刺激RAD51 活性主要是來自於穩定RAD51 核蛋白絲的生成。RAD51
是ssDNA 依賴的ATP 水解酵素。ATP 結合會促進RAD51 核蛋白絲的形成和股交換活性。然而,由於ATP 水解和低的ADP 釋放速率,會使有活性的ATP 結合的RAD51 核蛋白絲變成沒有活性的ADP結合的RAD51 核蛋白絲。我們進一步發現,SWI5-SFR1 會促進RAD51 核蛋白絲的ADP 釋放。這代表SWI5-SFR1 可以協助RAD51 核蛋白絲處在有活性的ATP 結合態。此外,我們也更進一步闡明SWI5-SFR1 和RAD51 交互作用的模式和區域。我們發現SWI5-SFR1 主要是跟多聚體RAD51 有交互作用。另外,我們也找到SWI5 的F83 和L85 對於和RAD51的交互作用是必要的。更重要的是,對於刺激RAD51 的活性是需要SWI5-SFR1和RAD51 的彼此交互作用。我們的研究結果闡釋了SWI5-SFR1 複合體刺激RAD51所主導的同源去氧核醣核酸的作用機轉。 Homologous recombination (HR) mediated by RAD51 recombinase eliminates DNA double-strand breaks in the genome. RAD51 forms a nucleoprotein filament on single-stranded DNA (ssDNA), termed the presynaptic filament, to initiate homologous recombination. Cytological studies in mammal indicate that SWI5 and SFR1 form a complex and participate in RAD51-mediated recombination repair. To decipher the mechanistic role of mammal SWI5-SFR1 complex in RAD51-mediated HR, we established the expression and the purification procedure to obtain mouse SWI5-SFR1 protein complex. Our biochemical study showed that SWI5-SFR1 complex stimulates homologous DNA pairing by RAD51 and stabilizes the RAD51 presynaptic filament, demonstrating that the stimulation of RAD51 activity stems from the stabilization of RAD51 filament by SWI5-SFR1 complex. RAD51 is an ssDNA dependent ATPase. ATP binding promotes the formation of a functional RAD51 nucleoprotein filament and DNA stand exchange activity. However, owing to ATP hydrolysis and slow dissociation rate of ADP, active RAD51 filament is converted into an inactive RAD51-ADP-ssDNA filament. We further documented that SWI5-SFR1 acts by facilitating the release of ADP from the RAD51 presynaptic filament, indicating that SWI5-SFR1 helps maintain RAD51 presynaptic filament in its active ATP bound form. Furthermore, we determined the interaction mode and region between SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially interacts with oligomeric form of RAD51. Importantly, the residue F83 and L85 in C-terminal SWI5 of SWI5-SFR1 complex is essential for the interaction of RAD51. The interaction of SWI5-SFR1 to RAD51 is indispensable for stimulation of RAD51 activity. Our results thus provide the insight for the action mechanism of RAD51-mediated DNA exchange by SWI5-SFR1 complex. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49197 |
DOI: | 10.6342/NTU201603201 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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