請用此 Handle URI 來引用此文件：
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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲 | zh_TW |
| dc.contributor.advisor | Jing-Jer Lin | en |
| dc.contributor.author | 李敏暄 | zh_TW |
| dc.contributor.author | Min-Hsuan Li | en |
| dc.date.accessioned | 2021-07-10T22:06:17Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-09 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Reference
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77512 | - |
| dc.description.abstract | 端粒為含有較高比例鳥糞嘌呤的重複性序列，位於染色體的末端能幫助維持染色體的穩定性。端粒酶為一種核糖核蛋白(ribonucleoprotein)，可以在在端粒的末端進行延長以維持端粒的長度。調節端粒酶活性的主要機制之一是通過端粒相關蛋白(telomere-associated proteins)之間的相互作用，進而去影響端粒酶在端粒上的可及性(accessibility)。在本篇研究中，分析了兩種端粒相關蛋白Pif1和Cdc13在端粒酶活性中的作用。Cdc13為單股端粒序列的結合蛋白，Cdc13與單股端粒的結合會抑制端粒酶的活性。通過與端粒酶相關蛋白Est1的相互作用，端粒酶活性受到Cdc13抑制的現象可以獲得緩解，因此得知Cdc13與端粒末端的結合在調節端粒酶活性中扮演相當重要的角色。Pif1為5’到3’的解螺旋酶，能將端粒酶從端粒上移除導致端粒酶活性受到抑制。Cdc13和Pif1都參與調控端粒酶活性，然而這兩種蛋白質如何在端粒上協調彼此在調控端粒酶活性上的功能目前仍尚不清楚。為解決這個問題，我們利用凝膠電泳分析(electrophoretic-mobility shift assay, EMSA)以及單分子栓球實驗(single-molecule tether particle motion, TPM)兩種in vitro系統。我們成功地利用大腸桿菌系統以及sf21昆蟲細胞系統分別純化出Cdc13重組蛋白和Pif1重組蛋白。結果顯示Pif1能夠移除位於單股端粒序列上Cdc13，且Pif1移除Cdc13的活性需要ATP和解旋酶活性。此結果不僅與Pif1解旋酶擁有轉位酶活性(translocase activity)的概念一致，也提出了Pif1有助於Cdc13置換的可能，進一步提供了一個新的調節端粒酶活性的機制。 | zh_TW |
| dc.description.abstract | Telomeres are G-rich repetitive sequences that cap the ends of chromosomes and help to maintain chromosomal integrity. Telomerase is a ribonucleoprotein that synthesizes telomeric repeats to help maintaining the length of telomeres. Controlling the accessibility of telomerase to telomeres by the interaction of telomere-associated proteins is one of the primary regulatory mechanism of telomerase activity. In this study, the roles of two telomere-associated proteins, Pif1 and Cdc13, in telomerase activity is analyzed. Cdc13 is a single-stranded telomeric DNA binding protein. Binding of Cdc13 to telomeric DNA inhibits telomerase activity. Through the interaction with Est1, a telomerase associated protein, the telomerase inhibitory activity can be rescued. Therefore, binding of Cdc13 onto telomere end plays an important role in regulating telomerase activity. Pif1 is a ATP-dependent 5’ to 3’ helicase. It also removes telomerase from telomere to inhibit telomerase activity. Both Cdc13 and Pif1 are involved in regulating telomerase activity, however, it is not clear how these two proteins coordinate their function on telomeres. Here I used both electrophoretic-mobility shift assay (EMSA) and single-molecule tether particle motion (TPM) systems to address this question in vitro. Recombinant Cdc13 and Pif1 were expressed and isolated from sf21 insect cells and E. coli, respectively. I found Pif1 is capable of removing Cdc13 from telomeric DNA. The Cdc13-removing activity of Pif1 requires both ATP and helicase activity. The results are consistent with a general notion that a Pif1 helicase also processes translocase activity. Moreover, the results suggest that Pif1 might contribute to Cdc13 displacement to further regulate telomerase activity. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T22:06:17Z (GMT). No. of bitstreams: 1 ntu-107-R05442014-1.pdf: 2475043 bytes, checksum: 1ab9cab7b54367a75687d8e9836dae72 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審訂書
謝辭 i 中文摘要 ii Abstract iii Contents v List of Tables vi List of Figures vi Introduction 1 Materials and Methods 9 Results 30 Discussion 40 Reference 46 Tables 51 Figures 54 | - |
| dc.language.iso | en | - |
| dc.subject | 端粒 | zh_TW |
| dc.subject | 端粒結合蛋白 | zh_TW |
| dc.subject | 端粒?活性調控 | zh_TW |
| dc.subject | telomerase activity | en |
| dc.subject | telomere | en |
| dc.subject | telomere-associated protein | en |
| dc.subject | Cdc13 | en |
| dc.subject | Pif1 | en |
| dc.title | 探討端粒結合蛋白Pif1和Cdc13的交互作用及在端粒酶活性調控上扮演的角色 | zh_TW |
| dc.title | Investigating the regulation of telomerase activity by telomere-associated proteins, Pif1 and Cdc13 in vitro | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 鄧述諄;冀宏源 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | 端粒,端粒結合蛋白,端粒?活性調控, | zh_TW |
| dc.subject.keyword | telomere,telomere-associated protein,Cdc13,Pif1,telomerase activity, | en |
| dc.relation.page | 64 | - |
| dc.identifier.doi | 10.6342/NTU201803440 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-15 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
| 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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| ntu-106-2.pdf 未授權公開取用 | 2.42 MB | Adobe PDF |
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