DHX8鄰酸化在DNA損傷反應中所扮演的角色

Abstract

DNA損傷反應，一直以來是醫學界以及我們實驗室所關切的議題，在維持基因體穩定性、細胞週期的檢查、程序性死亡等等，都需要依靠著精確的DNA損傷反應來協助我們。並針對路徑中更細節性的探討與研究，包含參與在其中的蛋白質所扮演的角色，會對DNA損傷反應有什麼樣的影響，以及其本身回應DNA損傷時，自身反應所產生的轉譯後修飾，我們實驗室主要針對探討相撲蛋白修飾化、磷酸化對於參與在DNA損傷反應路徑中的蛋白質進行研究。 在過去，實驗室以給予細胞紫外後收免疫沈澱，觀察到了DHX8在S460以及T554的這兩個磷酸位點的磷酸化程度會上升，因此指出了DHX8的磷酸化對於DNA損傷反應可能扮演著重要的角色，這也是連結DHX8對於DNA損傷反應的相關研究的接口。 除了在給予細胞紫外線損傷外，我也發現了在給予Camptothecin、Doxorubcin在其他種類的損傷下DHX8磷酸化程度會上升，顯示出了DHX8的磷酸化在DNA損傷當中所扮演的重要性。 而在本篇論文中，主要是探討DHX8的磷酸化，包含希望能找到DHX8的主要的磷酸化位點，因此想藉由實驗來指出這兩個位點對於DHX8回應DNA damage的重要性，並想知道若發生磷酸化位點的缺陷，會對於DNA損傷反應產生什麼影響，因此建構了DHX8-S460A、DHX8-T554A、DHX8-2A下去做磷酸化現象的研究，而從實驗結果，似乎可以看到S460在DHX8的磷酸化扮演著影響的角色，並且在T554這個位點若發生缺陷則會對細胞產生生長上的影響

The DNA damage response (DDR) has always been a concern in the medical community and our laboratory. Maintaining genomic stability, cell cycle checkpoints, programmed cell death, and more, all rely on precise DDR assistance. Exploring the finer details of the pathway and studying the roles played by proteins involved in it, including the effects on DDR and the translational post-translation modifications (PTMs) generated by its own response to DNA damage, our laboratory primarily focuses on investigating SUMOylation and phosphorylation of proteins involved in the DDR pathway. In the past, our laboratory observed an increase in phosphorylation levels at the S460 and T554 phosphorylation sites of DHX8 after exposing cells to Ultraviolet (UV) radiation, indicating that phosphorylation of DHX8 may play an important role in DNA damage response (DDR). This also serves as a link to relevant research on DHX8 and DDR. In addition to UV-induced DNA damage, I also found that phosphorylation of DHX8 increased under other types of damage induced by Camptothecin and Doxorubicin, demonstrating the importance of DHX8 phosphorylation in DDR. In this thesis, the main focus is on exploring the phosphorylation of DHX8, including identifying the main phosphorylation sites of DHX8. Therefore, experiments were conducted to demonstrate the importance of these two sites in DHX8's response to DNA damage, and to understand the effects of defects in phosphorylation sites on DDR. DHX8-S460A, DHX8-T554A, and DHX8-2A mutants were constructed for phosphorylation studies. From the experimental results, it seems that S460 plays a role in the phosphorylation of DHX8, and defects at the T554 site can affect cell growth.