2019-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/652512摘要：人類的癌細胞中，有部分的癌細胞是靠非端粒&#37238;的機制去延長端粒，以達到無限繁殖的能力，目前認為這個機制(Alternative Lengthening of Telomeres，簡稱ALT)是利用染色體端粒DNA的互換來延長端粒，這個機制是如何調控的還尚未釐清。最近發現在靠近端粒的附近有非編碼的RNA被轉錄出來，這個編碼的RNA稱之為TERRA，TERRA會跟端粒的DNA形成雜交，此計畫的目的在於研究TERRA 如何調控ALT及如何參與在染色體端粒DNA的互換，了解其機制後期望可發展出殺死ALT癌細胞的治療方法。我們先利用iDRiP的方法，來找出在ALT癌細胞中與TERRA結合的蛋白質，結果發現許多參與在DNA損傷與修復機制的蛋白可與TERRA作用。另外，我們利用大量表現RNase H來降解TERRA與端粒的DNA形成的雜交，結果顯示端粒聚集的現象降低，且端粒DNA縮短，推測TERRA與端粒的DNA形成的雜交可以幫助端粒的聚集，進而幫助染色體端粒DNA的互換來延長端粒DNA。綜合目前的研究結果我們提出一個假說，認為TERRA會與參與在DNA損傷與修復機制的蛋白作用來幫助ALT癌細胞端粒的延長。 <br> Abstract: A fraction of human cancer cells depends on a mechanism called alternative lengthening of telomere (ALT) to overcome telomere loss to reach immortality. It is commonly accepted that this mechanism is achieved by the homologous recombination among telomeres. Telomeric ends actively synthesize a heterogeneous population of long noncoding RNAs, which contain telomeric repeats, named “TERRA”. TERRA forms RNA–DNA hybrid structures (R-loops) at telomeres in human cancer cells. The unresolved TERRA:telomere hybrids (TERRA R-loops) can induce genome instability by impairing DNA replication and promoting recombination in budding yeast. However how TERRA involves in human ALT tumor remains unclear. This project is to study whether TERRA is involved in recombination for alternative lengthening of telomeres (ALT) and how it participates the recombination events in ALT cancer cells. The long-term goal is to target ALT pathway and develop a therapeutic method for cancer therapy. To investigate the role of TERRA in ALT mechanism, we performed iDRiP (identification of direct RNA-interacting proteins), a method that captures specific RNA’s interacting protein. We found a set of DNA damage proteins that interact with TERRA in U2OS cells. We eliminated TERRA R-loops by overexpression of RNase H in U2OS cells. We demonstrated that the level of telomere clustering was significantly decreased and the telomere length was shortened after RNase H overexpression. These results imply that TERRA R-loops promote homologous recombination via increasing telomere pairing. We hypothesize that DNA damage responsive proteins work with TERRA at R-loops to direct the DNA repair system to extend telomere length in human ALT cancer cells.非編碼RNA端粒染色體互換癌症TERRAnon-coding RNAtelomereR-loopsrecombinationalternative lengthening of telomeres cancer