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標題: | 探討Nucleostemin之生化特性及其於DNA複製壓力反應中之功能性角色 Investigating the Biochemical Characteristics of Nucleostemin and Its Functional Role in Replication Stress Response |
作者: | Yi-Hsuan Chang 張逸軒 |
指導教授: | 冀宏源(Hung-Yuan Chi) |
關鍵字: | 複製壓力,去氧核糖核酸雙股斷裂,Nucleostemin,RAD51,同源重組, Replication stress,DNA double-strand breaks,Nucleostemin,RAD51,Homologous recombination, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 去氧核糖核酸複製(DNA replication)為細胞分裂(cell division)能順利進行之重要步驟。然而,在去氧核糖核酸複製的過程中,複製叉(replication fork)的行進會因為各種威脅而受到阻礙,導致停滯複製叉(Stalled fork)的形成,甚至進而使崩塌複製叉(Collapsed fork)出現,造成去氧核糖核酸雙股斷裂(DNA double-strand breaks, DSBs)的產生,使細胞遭受複製壓力(replication stress)。增殖細胞(proliferating cells)必須進行大量之去氧核糖核酸複製,因此,其必須利用複製壓力反應(replication stress response)以維持基因體穩定性。Nucleostemin (NS)為一種特別於增殖細胞中高度表現的蛋白質。NS於近期被發現可能為參與複製壓力反應機制中的重要成員之一。NS的缺失會造成去氧核糖核酸雙股斷裂累積於S期(S-phase)的細胞中。另外,NS的缺失會造成去氧核糖核酸雙股斷裂修復蛋白RAD51聚集至去氧核糖核酸斷裂處之能力受到顯著影響。然而,NS於複製壓力反應中行使其功能之詳細機制,目前仍屬未知。
在此研究中,我們分別透過細胞培養以及生物化學實驗,來探討NS於複製壓力反應中的功能性角色。首先,我們透過去氧核糖核酸纖維實驗(DNA fiber assay),發現NS的缺失會造成停滯複製叉的重啟效率降低。同源重組(homologous recombination, HR)為崩塌複製叉的重要修復機制,透過同源重組報導實驗(HR reporter assay),我們發現NS的缺失使細胞的同源重組效率顯著降低。另外,我們建立了NS重組蛋白之表達及純化系統以進行生化實驗。藉由電泳遷移實驗(electrophoretic mobility shift assay, EMSA),我們發現NS對於複製叉之結合能力高於雙股(double-stranded DNA, dsDNA)或是懸伸去氧核糖核酸(overhang DNA)。另一方面,核酸酶保護實驗(nuclease protection assay)的結果顯示,NS可高度保護單股(single-stranded DNA, ssDNA)去氧核糖核酸免於核酸內切酶(endonuclease)之降解。此外,我們發現NS也具有保護單股/雙股去氧核糖核酸接合點(ssDNA/dsDNA junction)之能力。根據親和性沈降實驗(affinity pull-down assay),我們證實NS與RAD51之間存在直接且具物種專一性之蛋白質交互作用關係。綜合以上結果,我們推測NS參與於停滯及崩塌複製叉之重啟,並可能透過複製叉結合能力以及DNA保護能力,協同RAD51共同於複製壓力反應中行使功能。 DNA replication is a crucial step for proliferating cells to faithfully duplicate the genome during cell cycle. Nevertheless, collapsed replication forks will lead to DNA double-strand breaks (DSBs) and genomic instability. Nucleostemin (NS), a proliferating cell-enriched protein, has been identified as a key regulator for repairing replication-induced DNA damage. Loss of NS accumulates DSBs especially in S-phase cells. More importantly, NS regulates the recruitment of RAD51, an essential repair protein for DSBs and replication stress, to replication-induced DNA damage sites. However, the underlying mechanism for how NS functions in replication stress response remains largely unknown. Here, we report that lack of NS impedes the restart of stalled forks via DNA fiber assay. Moreover, NS depletion causes impaired HR efficiency which is essential for collapsed fork restart. With purified recombinant NS protein, we demonstrate that NS shows DNA binding preference for replication fork substrates over double-stranded or tailed DNA substrates. Using nuclease protection assay, we find that NS strongly protects ssDNA from endonuclease degradation. We further show NS also protects the DNA junction in overhang substrates. By pull-down assay, we demonstrate direct protein-protein interaction between NS and RAD51. Our results implicate that NS have important role in stalled and collapsed fork restart and may functions mechanistically through its fork binding preference and DNA protection activity by coordinated action with RAD51. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72128 |
DOI: | 10.6342/NTU201803116 |
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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