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標題: | 研究重組酵素結合去氧核糖核酸之調控 Studying the regulations of recombinases on DNA filament assembly |
作者: | Hao-Yen Chang 張皓衍 |
指導教授: | 冀宏源(Hung-Yuan Chi) |
關鍵字: | 同源重組,DMC1,RAD51,ATP 水解,小頭症蛋白 MCPH1, Homologous recombination,DMC1,RAD51,ATP hydrolysis,Microcephalin 1, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 當同源重組酵素RAD51透過同源重組修復DNA 雙股斷裂時,透過調控RAD51的ATP水解使得RAD51保持在ATP結合的活化態以穩定RAD51的核蛋白絲,對後續的雙股交換具有增強的作用。然而,對於減數分裂時期的同源重組酵素DMC1而言,調控ATP水解乃至於DMC1的核蛋白絲穩定,以及後續的雙股交換之間的關聯性仍是未知。為了探討這三者的關聯性,我們建立了蛋白表現及純化的步驟,得到人類DMC1正常蛋白與D317K點突變蛋白。經由我們的生化研究指出,在保有ATP結合的能力下,ATP水解抑制會促進DMC1核蛋白絲的穩定;然而,我們發現DMC1的雙股交換能力並未隨之提升,對於DMC1而言,單純ATP水解抑制並不能得到活化的DMC1核蛋白絲,顯示了減數分裂的同源重組酵素DMC1與同源重組酵素RAD51有相當大的不同。除了調控水解之外,我們也有興趣從疾病關聯性來尋找影響RAD51的核蛋白絲的調控因子。小頭症蛋白被發現與RAD51在細胞內有效結合DNA受損部位有關;因此,我們假設小頭症蛋白透過穩定RAD51的核蛋白絲來協助基因修補。為了驗證假設,我們首次建立了小頭症蛋白表現及純化的方法。透過生化研究顯示,我們意外發現小頭症蛋白具有DNA結合的能力,並且能直接結合RAD51;更重要的,透過不同研究方法,我們證明小頭症蛋白能直接穩定RAD51的核蛋白絲。 During repairing DNA double-strand breaks by RAD51-mediated homologous recombination, the presynaptic filament is maintained by regulating ATP hydrolysis of RAD51 to stay in ATP-bound form, which enhances the subsequent strand exchange. However, for meiotic specific homologous recombinase DMC1, the association between regulation of ATP hydrolysis and even nucleoprotein stabilization of DMC1, and subsequent DNA exchange is still unknown. To explore this issue, we established the protein expression and purification to obtain human DMC1 wild-type and D317K point mutant proteins. Through our biochemical studies, ATP hydrolysis inhibition promotes the stability of DMC1 presynaptic filaments while retaining ATP binding capacity; however, we found that DNA strand exchange capacity of DMC1 did not increase. For DMC1, simply ATP hydrolysis inhibition did not result in the activation of DMC1 nucleoprotein filaments, indicating that the meiotic homologous recombinase DMC1 is quite different from the homologous recombinase RAD51. Except for regulating ATP hydrolysis, we are also interested in finding the accessory factors regarding stabilizing RAD51 presynaptic filaments from disease association. Microcephalin 1 (MCPH1) had been found to be involved in the efficient binding of RAD51 to damaged sites in cells; therefore, we hypothesized that MCPH1 stabilizing RAD51 presynaptic filaments. To validate the hypothesis, we first established a method for expression and purification of MCPH1 protein. Through biochemical studies, surprisingly, we have discovered that MCPH1 protein has the ability to bind DNA and can directly bind to RAD51. Importantly, through different approaches, we have demonstrated that MCPH1 can stabilize presynaptic filaments of RAD51. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70374 |
DOI: | 10.6342/NTU201803267 |
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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