探討Cdc13及Pif1在端粒酶活性調控上所扮演的角色

Abstract

端粒是位於真核生物染色體末端的重複DNA序列結構，可以保護染色體末端不被分解，以維持染色體穩定性。由於聚合酶的限制，端粒會隨著染色體複製而越來越短，使細胞老化，細胞則可以使用端粒酶進行端粒延長以延遲老化現象發生。酵母菌的端粒酶是由反轉錄酶Est2及RNA分子TLC1組成，會利用自身TLC1 RNA當作引子延長端粒，而端粒酶活性會受到許多端粒相關蛋白的調控。Pif1是一個解螺旋酶，會坐落在單股DNA區域以5’端到3’端的方向將雙股解開。先前報導指出Pif1會透過將端粒酶從端粒末端移除而抑制端粒酶活性，在實驗室之前的研究中，我們發現了Cdc13會防止Pif1移除之單股DNA重新黏回受質，暗示著Cdc13可能會與Pif1一同調控端粒酶活性。有趣的是在引子延伸實驗當中，我發現了Pif1會增加端粒酶活性，此外，Cdc13會防止被Pif1移除之端粒酶再度結合至新的受質上面進行延伸，因而抑制端粒酶活性。在引子延伸實驗中，由於靈敏度及受質與端粒酶濃度比的限制，大部分看到的產物都是端粒酶進行一次循環延伸的產物，透過與台大化學系李弘文老師的合作，我們以布朗運動原理建立了單分子拴球實驗，應用於端粒酶活性檢測。透過單分子拴球實驗，我們可以觀察到端粒酶進行多次延伸循環產生之產物，另外，我們也發現了Cdc13會抑制端粒酶進行多次循環延伸反應。在本篇論文中，我們分別利用引子延伸實驗及單分子拴球實驗探討了Cdc13及Pif1對於端粒酶活性的調控。在未來，我們也會進一步將單分子拴球實驗應用於探討其他端粒結合蛋白是如何調控端粒酶活性，為端粒酶調控提供更多可能的機制。

Telomeres are regions of repetitive DNA sequences at the ends of eukaryotic linear chromosomes to protect chromosomes from deterioration. Due to the end replication problem, telomeres get shorter upon each cell division that lead cells to enter senescence. Telomerase has an enzymatic activity that utilizes a RNA molecule as template to elongate telomeres. Pif1 is a single-stranded DNA-dependent helicase which unwinds DNA duplexes in a 5’ to 3’direction. Pif1 was shown to inhibit telomerase activity through removing it from telomeres. Previously, we found that telomere binding protein, Cdc13, prevents the re-association of the Pif1-unwound DNA to telomeres, implicating that Cdc13 might collaborate with Pif1 to modulate telomerase activity. Using primer extension assay, I found that Cdc13 indeed prevents Pif1-removed telomerase from re-loading to new substrates. Due to the sensitivity and the high concentration ratio of DNA substrate to telomerase, primer extension assay is limited to observe mostly single-run products. Through the collaboration with Dr. Li HW (Department of Chemistry, National Taiwan University), we have established a single-molecule assay using tethered particle motion (TPM) to evaluate telomerase activity. TPM is based on the correlation of DNA length with the Brownian motion of a tethered particle. The assay enables the observation of telomerase extensions at the single molecule level. Using the assay, I found that Cdc13 inhibits multiple-run reactions of telomerase, consistent with the results from biochemical analyses. Thus, the single-molecule TPM approach can be used to investigate how other telomere-associated proteins regulate telomerase activity in the future.