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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Pang-Yen Wang | en |
| dc.contributor.author | 王邦硯 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:48:29Z | - |
| dc.date.available | 2022-08-31 | |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-14 | |
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M., Stretching DNA with optical tweezers. Biophys. J. 1997, 72 (3), 1335-1346. Odijk, T., Stiff Chains and Filaments under Tension. Macromolecules 1995, 28 (20), 7016-7018. Bockelmann,U.;Essevaz-Roulet,B.;Heslot,F., Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Phys. Rev. Lett. 1997, 79, 4489-4492. Smith, S. B.; Cui, Y.; Bustamante, C., Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 1996, 271 (5250), 795-799. Koch, S. J.; Shundrovsky, A.; Jantzen, B. C.; Wang, M. D., Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix. Biophys. J. 2002, 83 (2), 1098-1105. Manosas, M.; Spiering, M. M.; Ding, F.; Bensimon, D.; Allemand, J.-F.; Benkovic, S. J.; Croquette, V., Mechanism of strand displacement synthesis by DNA replicative polymerases. Nucleic Acids Res. 2012, 40 (13), 6174-6186. Kemmerich, F. 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J., Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress. Mol. Cell 2012, 47 (3), 396-409. Kile, A. C.; Chavez, D. A.; Bacal, J.; Eldirany, S.; Korzhnev, D. M.; Bezsonova, I.; Eichman, B. F.; Cimprich, K. A., HLTF's Ancient HIRAN Domain Binds 3' DNA Ends to Drive Replication Fork Reversal. Mol. Cell 2015, 58 (6), 1090-1100. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78265 | - |
| dc.description.abstract | 在細胞進行DNA 複製的過程中，當複製叉遇到特定的序列或是DNA 損傷時， 可能會造成複製叉的停滯並脫落，此時裸露出的DNA 極其脆弱，可以造成基因體 的不穩定性，細胞可以利用複製叉的回溯將停滯的複製叉重啟成可再次進行複製 的DNA 構型。進行DNA 回溯的第一步，是黏和DNA 叉中裸露的兩個互補股。 人類細胞中SNF2 家族的黏和酶具有這個功能，其中SMARCAL1 已被報導在複製 叉回溯扮演主要角色，但是其黏和反應的機制尚不明確。由於兩個DNA 互補股的 黏合反應，在一般平均的生化實驗中很難量測，本論文利用單分子光鉗技術，直接 研究SMARCAL1 在DNA 叉上的黏和反應及其動力學。實驗結果發現在含有ATP 的狀態下，SMARCAL1 的黏和反應與DNA 叉所受的張力相關，在15 pN 的張力 下，SMARCAL1 的黏和反應明顯被抑制。將張力降至7 pN 時，則可偵測到明顯 的黏和反應的產物。比較DNA 複製中引導股缺口及延遲股缺口這兩種停滯複製叉 結構發現，SMARCAL1 在延遲股缺口的黏和反應性較好，但是其黏和的長度相當， 代表其兩者黏和機制可能相同。 | zh_TW |
| dc.description.abstract | During DNA replication, polymerases, from time to time, encounter DNA lesion or DNA structure that stall the replication fork and potentially lead to the replication fork collapse. Stalled or collapsed replication forks must be properly restarted to resume replication for cell vivability. Fork reversal has been suggested to be an efficient way to restart replication. The first step of fork reversal is to anneal the exposed, two complementary DNA strands. In human cells, this task can be done by SNF2 family annealing helicases, and SMARCAL1 has been reported to play a major role in fork reversal. The functional mechanism associated with the SMARCAL1-mediated fork reversal is not clear. DNA strand annealing activity is challenging to monitor in conventional, ensemble-based biochemical assays. This study utilizes single-molecule optical tweezers to directly monitor the annealing activity of the SMARCAL1 on the DNA fork in real-time. Our results show the annealing activity of SMARCAL1 depends on the force applied. In the presence of 15 pN tension, no apparent annealing activity is seen. Lowering the force to 7 pN, much more annealing events are observed. We compared two replication forks with structure containing either a lagging-strand gap or a leading-strand gap. SMARCAL1 has preference on the lagging-strand gap structure, as the frequency of annealing events is higher. However, both fork structures have similar processivity and translocation rate, likely reflective of the same annealing mechanism. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:48:29Z (GMT). No. of bitstreams: 1 U0001-1108202022570000.pdf: 4599082 bytes, checksum: 17ea9a3cdd20bcdf7054b5864cdc07b1 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 # 致謝 i 摘要 ii Abstract iii CONTENTS iv 圖目錄 vi 表目錄 viii 第1章 緒論 1 1.1 複製叉回溯反應 (replication fork reversal) 1 1.2 人類細胞的複製叉的回溯反應 3 1.3 DNA纖維絲實驗決定複製叉回溯因子 5 1.4 DNA黏和酶SMARCAL1作為複製叉重塑的功能 6 1.5 力鉗技術研究DNA黏和酶實驗 8 1.6 研究動機 10 第2章 實驗方法與設計 11 2.1 SMARCAL1蛋白得純化以及保存 11 2.2 DNA 基質合成 11 3281 bp 全雙股DNA 11 Unzipping DNA的製作 12 從unzipping DNA延伸之DNA結構 15 DNA引子序列 16 2.3 實驗操作方式 17 製備光鉗實驗用玻片 17 第3章 光鉗系統實驗設計 18 3.1 光鉗基本原理 18 3.2 光鉗系統之光路架設 20 3.3 雷射力常數校正 22 靈敏度及彈力常數校正 22 雷射中心高度校正 24 3.4 量測DNA分子之長度與硬度資訊 25 3.5 Unzipping DNA基本性質測量 27 Unzipping DNA 力-伸長量曲線 27 Unzipping DNA 限制酶結合位觀察實驗 29 被動式力鉗實驗(Passive mode assay) 31 被動式定力鉗實驗(passive force clamp assay) 33 第4章 實驗結果與討論 34 4.1 被動式力鉗實驗(Passive mode assay) 34 4.2 被動式力鉗觀察hRPA單股DNA結合反應 35 4.3 峰值尋找演算法 37 4.4 以被動式力鉗觀察SMARCAL1的黏和反應 39 被動式力鉗無法明確分辨出SMRCAL1之反應作用 39 4.5 被動式定力鉗實驗 (Passive force clamp assay) 42 4.6 以被動式定力鉗觀察SMARCAL1的黏和反應 43 SMARCAL偏好黏和延遲股缺口 43 SMARCAL1在延遲股缺口有兩種黏合模式 45 第5章 結論與未來展望 46 5.1 結論 46 5.2 未來展望 47 參考文獻 49 附錄 53 附錄一 單分子栓球玻片製作方式 53 附錄二 表面鍊合酶素修飾聚苯乙烯球反應 54 | |
| dc.language.iso | zh-TW | |
| dc.subject | 黏和反應 | zh_TW |
| dc.subject | 單分子光鉗技術 | zh_TW |
| dc.subject | 複製叉回溯 | zh_TW |
| dc.subject | fork reversal | en |
| dc.subject | optical tweezers technique | en |
| dc.subject | annealing activity | en |
| dc.title | 以單分子光鉗方式觀察 SMARCAL1 的複製叉回溯 | zh_TW |
| dc.title | Direct observation of SMARCAL1-mediated fork reversal using optical tweezers | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 冀宏源(Hung-Yuan Chi),詹迺立(NEI-LI CHAN),廖泓鈞(Hung-Jiun Liaw) | |
| dc.subject.keyword | 單分子光鉗技術,複製叉回溯,黏和反應, | zh_TW |
| dc.subject.keyword | fork reversal,optical tweezers technique,annealing activity, | en |
| dc.relation.page | 54 | |
| dc.identifier.doi | 10.6342/NTU202003022 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-17 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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