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

蘇綸勤(Guan-Chin Su)

指導教授 ： 冀宏源

國立臺灣大學/生命科學院/生化科學研究所/博士(2016年)

https://doi.org/10.6342/NTU201603201

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。

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

並列摘要

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.

並列關鍵字

Homologous recombination ； DNA double-strand breaks ； RAD51 presynaptic filament ； SWI5-SFR1 ； ATP hydrolysis ； ADP release

參考文獻

1. Ciccia, A. and Elledge, S.J. (2010) The DNA Damage Response: Making It Safe

2. Lord, C.J. and Ashworth, A. (2012) The DNA damage response and cancer

therapy. Nature, 481, 287-294.

recombination and human health: the roles of BRCA1, BRCA2, and associated

proteins. Cold Spring Harb Perspect Biol, 7, a016600.