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標題: | 探討在DNA損傷參與的熱休克蛋白基因表現中,DNA-PK及拓樸異構酶扮演的角色 Investigate on the roles of DNA-dependent protein kinase and topoisomerase in the gene expression of heat shock protein 70 accompanied with DNA damage |
作者: | 李奕萱 I-Hsuan Lee |
指導教授: | 李財坤 Tsai-Kun Li |
關鍵字: | 熱休克蛋白,拓樸異構?, heat shock protein,topoisomerase, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 在轉錄的起始 (initiation) 與延伸 (elongation)的過程中,有很多因子會進行調控,除了一般熟知的轉錄因子之外,一些DNA損傷修復相關蛋白也會參與在訊號引起的轉錄啟動與延伸的調控。在賀爾蒙引起的基因轉錄例子中,在給予細胞賀爾蒙的刺激後,賀爾蒙刺激下游基因其啟動子的區域會有DNA雙骨斷裂的生成,同時DNA修復相關蛋白DNA-PK及PARP-1會聚集到DNA斷裂形成的位置,此外,第二型人類拓樸異構酶亞型 (Top2b) 在賀爾蒙刺激之後也會聚集到轉錄子的位置,根據此現象,我們認為在賀爾蒙調控的轉錄中,啟動子位置的DNA損傷反應會伴隨著轉錄啟動的發生,且會產生DNA雙股斷裂的Top2b會參與其中。除了賀爾蒙引起的轉錄之外,DNA損傷反應也發生在壓力刺激引起的轉錄中,先前的研究運用熱刺激的形式來分析DNA損傷反應和RNA聚合酶II (pol II) 釋放前進的關聯性,RNA pol II若成功從轉錄起始點(Transcription Start Site) 釋放前進,則可使轉錄順利進入下一個延伸的階段,而在給予細胞43℃的熱刺激之後,在熱休克蛋白基因的轉錄起始點會有磷酸化的變異組蛋白 (gH2AX) 生成,其可間接代表DNA雙股斷裂。根據這個現象,我們用熱刺激的方式來探討DNA-PK及Top2b在pol II 從TSS位置釋放前進中所扮演的角色,在給予細胞10分鐘43℃的熱刺激之後,pol II、DNA-PKcs、Ku80、Top2b皆會聚集到TSS附近的區域,同時gH2AX也會在TSS區域生成,此外,為了要探究DNA-PK在熱刺激後的活化,我們也確認DNA-PKcs不同的磷酸化位點,包含在DNA-PK自體磷酸化的絲胺酸2056位點 (S2056)及會被ATM激酶或DNA-PK本身磷酸化的蘇胺酸2609位點 (T2609),我們發現DNA-PKcs在熱刺激五分鐘後,會磷酸化在T2609的位點,接著DNA-PKcs在S2056的位點在熱刺激後10分鐘也會被磷酸化,磷酸化的DNA-PK代表DNA-PK不只有聚集到TSS附近位置,也代表DNA-PK會被ATM或是被自己的激酶作用活化。另一方面,在43℃熱刺激之後,給予細胞Top2的抑制劑 ICRF-193會使熱休克基因表現下降,我們可從此得知在轉錄延伸的過程當中需要Top2b的參與,總結,Top2b及DNA-PK會參與在pol II從TSS區域釋放進入延伸階段中,同時也在轉錄延伸中扮演正向的角色。 There are several factors involved in transcription regulation during initiation and elongation. In addition to general transcription factors, DNA damage response (DDR)-related proteins also participate in regulation of transcription activation and elongation in signal-induced transcription. In hormone-induced cases, transient DNA double stranded breaks (DSBs) occur at the promoter region of hormone-induced gene after treatment of hormone. Meanwhile, repair-related proteins DNA-dependent protein kinase (DNA-PK) and Poly [ADP-ribose] polymerase 1 (PARP-1) recruit to the same site along with the DSBs formation. Besides, topoisomerase IIbß(Top2b) also recruits to the same region after induction of hormone. Based on this phenomenon, we thought that DDR signaling is parallel to transcription activation in promoter region with the attendance of Top2b, which may induce DSBs in hormone-induced models. In addition to hormone-induced transcription, DDR signaling also occurs at the stress-induced transcription. Previous studies used heat shock model to analyze the relationship between DDR signaling and pol II pausing release, which is important for productive elongation. There is the signal of gH2AX, which can indirectly represent DSBs, at the region near transcription start site (TSS) of HSPA1B after induction of 43℃ heat shock. Based on this phenomenon, we used this heat shock model to explore the roles of DNA-PK and Top2b in regulation of pol II pausing release. After 10 minutes heat shock treatment at 43℃, pol II, DNA-PKcs, Ku80, Top2b all recruit to the region near TSS. The signal of H2AX also increase at the same site after 10 minutes heat shock. To demonstrate the activation of DNA-PKcs in this model, we also checked the different activated form of DNA-PKcs containing phosphorylated sites at S2056, which is autophosphorylation, and at T2609, which can be phosphorylated by itself or by ataxia-telangiectasia mutated (ATM) kinase. We found that DNA-PKcs is first phosphorylated at T609 at 5 minutes heat shock. Further, phosphorylation of DNA-PKcs at S2056 occurs after 10 minutes heat shock following phosphorylated cluster at T2609. The phosphorylated DNA-PK indicates that DNA-PK not only recruits to TSS region but also activates by itself or by ATM kinase. On the other hands, the gene expression of HSPA1B decreases in the treatment of Top2 inhibitor ICRF-193 on MCF-7 cells. We can conclude that Top2b is required for transcription elongation. Taken together, Top2b and DNA-PK may be involved in pol II pausing release at the region near TSS and play positive roles in transcription elongation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79114 |
DOI: | 10.6342/NTU201802381 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-11 |
顯示於系所單位: | 微生物學科所 |
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