DHX9解旋酶在基因體穩定性之角色

The role of DHX9 in genomic stability

林耿如(Keng-Ru Lin)

指導教授 ： 吳青錫

國立臺灣大學/醫學院/藥理學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202001528

摘要

DHX9為NTP-dependent DExH-box helicase，是一種能利用水解NTP所產生的能量，解開雙股RNA、雙股DNA、DNA/RNA雜合體等複雜結構的解旋酶。DHX9對許多細胞生理過程相當重要，包含RNA轉錄及轉錄後加工、DNA複製，以及近期發現維護基因體穩定性之功能。
ATM和ATR是DNA損傷訊息傳遞路徑中的兩個主要訊息傳遞激酶，他們會藉由磷酸化受質的SQ/TQ motifs調控DNA損傷修復。過去的磷酸化蛋白質體研究指出，DHX9的Ser321或Ser688位點會被ATM或ATR磷酸化，這暗示在DNA損傷後ATM或ATR可能會調控DHX9。因此，我首先要探討DHX9是否參與DNA損傷修復機制。實驗結果顯示沉默DHX9基因會減少ATR-Chk1訊息傳遞路徑的活化，但並不影響DNA合成、DNA雙股斷裂的產生和募集RPA到DNA損傷位置。細胞存活分析也顯示沉默DHX9會使癌細胞對ATR抑制更加敏感，暗示DHX9可能作用在ATR-Chk1訊息傳遞路徑。另外，透過蛋白質體分析，我找出許多會和DHX9交互作用的蛋白質，他們可能進而影響DHX9對ATR-Chk1訊息傳遞路徑的調控。藉由不同的基因毒性物質，我證實ATR會主導DHX9 Ser321位點的磷酸化，而沉默DHX9會在DNA損傷情況下使R-loops生成增加。進一步研究也顯示，在沒有基因毒性壓力情況下，表現DHX9的S321/688A phosphor (DS)或helicase/ATPase-dead (DE)突變型都會導致γH2AX及RPA pSer4/Ser8表現量上升。
總結來說，我認為DHX9會調控ATR-Chk1訊息傳遞路徑，且ATR對DHX9的調控為前饋控制。

關鍵字

DHX9 ； DNA損傷修復 ； DNA損傷訊息傳遞路徑 ； ATR-Chk1訊息傳遞路徑 ； R-loops

並列摘要

DHX9 is a NTP-dependent DExH-box helicase that can hydrolyze different NTPs to unwind RNA or DNA duplexes, DNA/RNA hybrids, and other more complex structures. DHX9 is important for many cellular processes including transcription, processing of RNA, DNA replication, and a recent emerging role in the maintenance of genomic stability.
ATM and ATR are two major transducer kinases in DNA damage-signaling pathways, which coordinate the DNA damage response (DDR) via phosphorylation of substrates on SQ/TQ motifs. Previous phosphoproteomic studies have demonstrated that Ser321 and Ser688 of DHX9 are phosphorylated by ATM/ATR, suggesting the regulation of DHX9 by ATM/ATR in response to DNA damage. Here I first investigated whether DHX9 participates in the DDR. The depletion of DHX9 results in reduced activation of the ATR-Chk1 signaling pathway, whereas DNA synthesis, the formation of DNA double-stranded breaks (DSBs), and the recruitment of RPA to sites of DNA damage are not affected. Cell viability assay further shows that cancer cells depleted of DHX9 are hypersensitive to ATR inhibitor suggesting DHX9 functions in the ATR-Chk1 signaling pathway. Furthermore, proteomic analysis reveals numerous DHX9 interactors that may potentially account for the DHX9 regulated ATR-Chk1 signaling pathway. Next, I confirmed that DHX9 is mainly phosphorylated at Ser321 by ATR in response to different types of genotoxic agents. The knockdown of DHX9 leads to an increased formation of R-loops upon DNA damage. Further investigation shows that expressing S321/688A phosphor (DS) or helicase/ATPase-dead (DE) mutants result in increased γH2AX and RPA pSer4/Ser8 in the absence of genotoxic stress.
Collectively, I proposed that DHX9 regulates the ATR-Chk1 signaling pathway and the existence of a feed-forward loop of ATR-DHX9 circuitry.

並列關鍵字

DHX9 ； DNA damage response ； DNA damage-signaling pathways ； ATR-Chk1 signaling pathway ； R-loops

參考文獻

1. Rouse, J., and Jackson, S.P. (2002). Interfaces between the detection, signaling, and repair of DNA damage. Science 297, 547-551.

2. Jackson, S.P., and Bartek, J. (2009). The DNA-damage response in human biology and disease. Nature 461, 1071-1078.

3. Smith, J., Tho, L.M., Xu, N., and Gillespie, D.A. (2010). The ATM–Chk2 and ATR–Chk1 pathways in DNA damage signaling and cancer. Advances in Cancer Research 108, 73-112.

4. Kinner, A., Wu, W., Staudt, C., and Iliakis, G. (2008). γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research 36, 5678-5694.

5. Beishline, K., and Azizkhan-Clifford, J. (2014). Interplay between the cell cycle and double-strand break response in mammalian cells. Methods in Molecular Biology 1170, 41-59.