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標題: | 以單分子光鉗方式觀察 SMARCAL1 的複製叉回溯 Direct observation of SMARCAL1-mediated fork reversal using optical tweezers |
作者: | Pang-Yen Wang 王邦硯 |
指導教授: | 李弘文(Hung-Wen Li) |
關鍵字: | 單分子光鉗技術,複製叉回溯,黏和反應, fork reversal,optical tweezers technique,annealing activity, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78265 |
DOI: | 10.6342/NTU202003022 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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