利用生物物理與生化分析方法探討反轉複製叉家族蛋白於跨越 DNA 損傷的作用機制

Biophysical and Biochemical Approaches on How Fork Reversal Enzymes Function in DNA Lesion Bypass Mechanism

王冠期(Kuan-Chi Wang)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101974

摘要

進行 DNA 複製過程中，損傷的DNA 模板會導致 DNA 複製叉停滯。人體反轉複製叉酵素 (SMARCAL1、ZRANB3、HLTF) 可將停滯的 DNA 複製叉，轉換成雞爪形狀之反轉複製叉，以保護複製叉結構及增加 DNA 穩定性，為細胞爭取時間修復受損的 DNA。反轉複製叉形成過程包括兩條新生股與原先模板股解除配對，兩條模板股相互配對，隨後新生股相互配對，以形成四股 DNA 交叉。然而目前對於反轉複製叉酵素如何催化複製叉反轉機制，仍尚未釐清，本論文利用生化方法與單分子螢光共振能量轉移技術，探討反轉複製叉酵素的作用機制。通過單分子螢光共振能量轉移實驗，我們驗證了三種人類反轉複製叉酵素皆可以有效地進行反轉複製叉反應，且作用速率相似。接著，利用生化解旋酶實驗發現三種反轉複製叉酵素皆具有解旋酶特性，可將 DNA 兩股分離，但各自特性不同: SMARCAL1 僅能解旋母股，而 ZRANB3, HLTF 能夠解旋前進與延遲股。利用 DNA 兩股的生化黏合實驗中不互補序列在不同的複製叉位點，發現 SMARCAL1 與 ZRANB3, HLTF 利用不同黏合機制，SMARCAL1 無法跨越位於三股 DNA 交叉的損傷位點，然而 ZRANB3 與 HLTF的反應性卻不受阻； ZRANB3 與 HLTF 黏合機制類似但卻有不同的序列不互補忍受度，這些結果讓我們定義出這些反轉複製叉酵素的特性，並為這些反轉複製叉酵素的作用機制提出基本的生化模型。

關鍵字

複製叉反轉 ； 解旋酶 ； SMARCAL1 ； ZRANB3 ； HLTF ； 單分子螢光共振能量轉移螢光共振能量轉移

並列摘要

During replication, stalled replication forks containing single-stranded DNA structure activates replication stress response and cause genome instability, cell death and diseases. A critical point of the replication stress response is to remodel stalled replication forks into reversed forks. Three human fork reversal enzymes: SMARCAL1, ZRANB3, HLTF have been identified to be essential in fork regression. Fork regression includes steps involving: two nascent strands unpairing from their parental templates, two parental strands re-annealing and two nascent strands annealing to form a four-way junction. However, mechanistic understanding of how these fork remodelers function is lacking. Here, we first used single molecule fluorescence resonance energy transfer experiments to monitor the reversed fork formation in realtime. These three fork reversal enzymes all catalyze fork reversal at similar rates. We also used biochemical helicase assay to demonstrate that all three fork reversal enzymes have helicase activity, but with different substrate preferences. SMARCAL1 can only unwind parental strands, while ZRANB3 and HLTF unwind two leading and lagging strands. Using replication forks with mismatched sequences at different locations, we found that SMARCAL1 use a different mechanism to bypass DNA lesion, comparing to ZRANB3 and HLTF. On the other hand SMARCAL1 can’t bypass lesion on 3 strand Junction while ZRANB3 and HLTF can efficiently bypass it. These results form the molecular base to our under standing of how these three fork reversal enzymes function.

並列關鍵字

reverse fork ； helicase ； SMARCAL1 ； ZRANB3 ； HLTF ； single molecule FRET

參考文獻

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