以單分子技術研究酵母系統中Pif1解旋酶調控端粒酶與鳥嘌呤-四股結構之機制

Using Single-Molecule Techniques to Investigate How ScPif1 Regulates Yeast Telomerase Activity and G-quadruplex Formation

李靜如(Jing-Ru Li)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.02421

摘要

端粒酶 (telomerase)，為一核酸蛋白複合體，負責延長真核細胞染色體尾端的端粒序列(telomere)。利用其核酸部分作為模板股，端粒酶能夠執行反轉錄的功能來延長染色體尾端的不斷重複的端粒片段。研究指出，Pif1解旋酶具有調控端粒酶的能力。我們利用單分子栓球實驗，直接地觀察到Pif1解旋酶從端粒末段將端粒酶移除，進而調控端粒酶的反應。首先，我們觀察到Pif1利用水解三磷酸腺苷 (ATP) 的能量來移除端粒酶，使得原本與端粒穩定結合的端粒酶，得以與新的端粒片段反應，導致整體被延長的端粒比例增加。在移除過程中，較長的單股去氧核醣核酸與高濃度的Pif1都能展現較好的移除效率，根據這些實驗結果，我們建立一簡單模型去解釋Pif1如何調控端粒酶活性以及端粒長度的機制。由於端粒末段富含GC的序列，可以形成鳥嘌呤-四股的特殊結構。因此，四股結構的形成可以影響基因表現以及端粒與端粒酶的作用。Pif1已被指出具有解開鳥嘌呤四股結構的能力，我們利用單分子栓球實驗直接觀察Pif1能有效解開鳥嘌呤四股結構，並接續解開下游的雙股核酸，指出Pif1的解旋活性及移除端粒酶性質，可以在細胞環境下用以調控具有鳥嘌呤四股結構的端粒長度與端粒酶活性。

關鍵字

端粒酶 ； 端粒 ； Pif1解旋酶 ； 鳥嘌呤-四股結構 ； 單分子栓球實驗

並列摘要

Telomerase, a ribonucleoprotein complex, is responsible for maintaining the telomere length at chromosome ends. Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3’-end single-stranded, repetitive telomeric DNA sequence. Pif1, a 5’-to-3’ helicase, has been suggested to regulate telomerase activity. We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional processes. This telomerase removal efficiency increases at longer ssDNA gaps and at higher Pif1 concentration. The enhanced telomerase removal efficiency by Pif1 at the longer single-stranded telomeric DNA suggests a way of how Pif1 regulates telomerase activity and telomere length. On the other hand, G-quadruplex is specific nucleic acids structure which is reported to be present in the promoter regions as well as telomeres with GC-rich sequence. The folding of G-quadruplex is implicated in regulating gene expression and telomere maintenance. Pif1 monomer is evidenced to be able to resolve the G-quadruplex structure using the “patrolling” mechanism without leaving the G-quadruplex sequence to keep unwinding the downstream duplex DNA. Here, we observed that Pif1 is capable of unwinding the dsDNA immediately following the G-quadruplex unwinding in the high Pif1 concentration. Our results suggest by resolving the G-quadruplex during replication and transcription, Pfi1 may play important roles not only in regulating telomere length but also in maintaining genome stability.

並列關鍵字

telomerase ； telomere ； Pif1 helicase ； G-quadruplex ； single molecule TPM

參考文獻

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