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標題: | 端粒酶缺乏之釀酒酵母之老化後期倖存者機制分析 Mechanistic Analysis of Post-senescent Survivors in Telomerase-deficient Saccharomyces cerevisiae |
作者: | 劉家君 Chia-Chun Liu |
指導教授: | 林敬哲 Jing-Jer Lin |
關鍵字: | 端粒,第二型倖存者,ALT,DNA錯配修復,TERRA,R-loop,Rad27,染色質重塑蛋白, telomere,ALT,mismatch repair,type II survivors,TERRA,R-loop,Rad27,chromatin remodeler, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 端粒對於維持染色體完整性至關重要,在DNA複製過程中的端粒縮短最會終導致細胞衰老。儘管大多數真核生物依賴端粒酶維持端粒長度,但一小部分細胞利用一種稱為 “alternative lengthening of telomeres” (ALT) 的重組機制恢復端粒長度。在酵母Saccharomyces cerevisiae中,缺乏端粒酶的倖存者通過同源重組恢復端粒長度,分為第一型型存活者 (Y' element複製,依賴RAD51路徑) 和第二型倖存者 (長而異質的TG序列,不仰賴RAD51)。先前的研究發現,“telomeric repeat-containing RNA” (TERRA) 和RNA-DNA hybrid在促進II型重組中扮演著重要角色。在本研究中,我們使用了兩種策略來篩選參與調控端粒重組的其他因子。在第一種策略中,我們篩選了具有核酸酶活性的蛋白質,並發現SAE2和RAD27的突變對tlc1細胞中的存活者形成產生影響。SAE2是人類CtIP的同源物,調控MRX (Mre11-Rad50-Xrs2) 複合物的內切酶活性。表達Sae2-D285P/K288P (缺乏Mre11調節活性) 無法拯救sae2 tlc1細胞中第二型端粒重組延遲形成的表型,表明Sae2通過調節MRX複合物的活性參與第二型端粒重組。Rad27是人類Fen1的同源物,參與移除岡崎片段的5' flap。缺乏RAD27會導致第二型倖存者提早產生。Rad27藉由切割RNA-DNA hybrid的5' flap,阻止TERRA和RNA-DNA hybrid在端粒上的累積,從而防止C-circle形成和第二型端粒重組。在第二種策略中,我們使用生物信息學和已發表的數據分析了蛋白作用網絡,發現mismatch repair (MMR) 因子和chromatin remodeler蛋白作為候選調節因子。MutSα (Msh2-Msh6) 和MutLα (Mlh1-Pms1) 的突變在tlc1細胞中會延遲第二型倖存者出現。儘管msh2 rad51 tlc1突變體仍然會產生第二型倖存者,這代表MMR對於第二型重組不是必需的,但MMR仍在抑制第一型重組和促進第二型重組中發揮作用。Chromatin remodeler中的INO80複合物突變體在我們的篩選中發現會促進第二型倖存者的形成。缺失INO80會降低tlc1細胞的細胞存活能力並加速端粒侵蝕。此外,ino80 tlc1細胞表現更高的TERRA表達量,表明INO80複合物在抑制TERRA表達方面發揮作用。 Telomeres are crucial for maintaining chromosome integrity, and their shortening during DNA replication leads to cellular senescence. While most eukaryotes rely on telomerase to maintain telomere length, a small fraction of cells employ a recombination-based mechanism called alternative lengthening of telomeres (ALT). In yeast, rare survivors lacking telomerase regain telomere length through homologous recombination, classified into type I survivors (amplified subtelomeric Y' elements, RAD51-dependent) and type II survivors (long heterogeneous TG sequences, RAD51-independent). Previous studies found that telomeric repeat-containing RNA (TERRA) and RNA-DNA hybrids play roles in promoting type II recombination. Here, we used two strategies to screen other factors involved in regulating telomere recombination. In the first strategy, we screened proteins with nuclease activities and identified SAE2 and RAD27 mutations affecting survivor formation in tlc1 cells. Sae2, the homolog of human CtIP, regulates the endonuclease activity of the MRX (Mre11-Rad50-Xrs2) complex. Expression of Sae2-D285P/K288P, a mutant with impaired Mre11 regulation activity, failed to rescue the late type II formation phenotype in sae2 tlc1 cells, indicating Sae2's contribution to type II recombination through regulation of MRX complex activities. Rad27, the homolog of human Fen1, participates in removing the 5' flap of Okazaki fragments. Depletion of RAD27 led to early formation of type II survivors. By cleaving the 5' flap of RNA-DNA hybrids, Rad27 inhibited TERRA and RNA-DNA hybrid accumulation on telomeres and preventing C-circle formation and type II recombination. In the second strategy, we analyzed the interaction network using bioinformatics and published data, identifying mismatch repair (MMR) factors and chromatin remodelers as candidate regulators. Mutations in MutSα (Msh2-Msh6) and MutLα (Mlh1-Pms1) exhibited a late onset of type II survivors in tlc1 cells. Although type II survivors still emerged in msh2 rad51 tlc1 mutants, indicating that MMR is not required for type II recombination, MMR played a role in inhibiting type I recombination and promoting type II recombination. Chromatin remodeler INO80 complex mutants promoted type II survivor formation in our screening. Depletion of INO80 decreased cell viability and accelerated telomere erosion in tlc1 cells. Additionally, ino80 tlc1 cells exhibited higher TERRA expression, suggesting that the INO80 complex played a role in inhibiting TERRA expression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90314 |
DOI: | 10.6342/NTU202302190 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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