探討ZRANB3寡聚態在複製叉反轉 及DNA解旋中的功能

Abstract

複製叉反轉是細胞因應複製壓力以維持基因體穩定性的重要調控機制。在人類細胞中，SNF2 家族的 DNA 移位酶包括 SMARCAL1、ZRANB3與HLTF皆參與此反應。ZRANB3蛋白已被證實能催化複製叉反轉，並具有類似於解旋酶活性，能解旋約 15 個鹼基長的新生股。對於多數解旋酶來說，其寡聚態狀態在酵素活性調控中扮演重要角色。先前研究透過光漂白實驗發現，單一分叉形DNA受質上可以容納大於一個的ZRANB3分子。然而，ZRANB3 在反應過程中的寡聚態狀態及其功能仍未被釐清。因此，本研究利用單分子螢光技術，結合光漂白分析與螢光共振能量轉移，探討 ZRANB3 在複製叉反轉及 DNA 解旋反應中的寡聚態行為與動力學特徵。結果顯示，單體或二聚體形式的ZRANB3 ，皆可有效催化複製叉反轉，且在催化反應期間ZRANB3的寡聚態狀態不會改變。當 RPA 結合於延遲股缺位時，單體與二聚體 ZRANB3 皆可移除 RPA 並維持與沒有RPA結合時的反應效率；相對地，前進股缺位上的 RPA 則會抑制整體反應，但不影響 ZRANB3 的寡聚態分布。在進行 RPA 移除實驗時，我們發現在高濃度條件下，ZRANB3 能夠解旋長度達 30 個鹼基的前進新生股。在濃度梯度實驗中，我們將ZRANB3的RPA移除速率與DNA解旋速率對ZRANB3的濃度做圖，結果顯示DNA解旋反應呈現S型曲線，而RPA移除反應則呈現雙曲線，兩者反應表現出不同的濃度依賴動力學特徵。此外，雖然隨著蛋白濃度上升，結合至 DNA 受質上的二聚體比例顯著增加，但若直接比較單體與二聚體的解旋反應，二聚體並未表現出較高的反應速率或效率。綜合上述結果，DNA解旋反應所觀察到的S型曲線可能與ZRANB3寡聚態分布與DNA結合行為不同有關，而非來自單一寡聚態在催化步驟上的差異。本研究從單分子層級探討 ZRANB3 寡聚態與其功能，進一步釐清複製叉反轉與 DNA 解旋的調控機制，並提供新的動力學觀點。

Fork reversal is a key mechanism that enables cells to respond to replication stress and maintain genome stability. In human cells, SNF2 family DNA translocases, including SMARCAL1, ZRANB3, and HLTF, participate in this process. ZRANB3 has been shown to catalyze fork reversal and exhibits helicase-like activity capable of unwinding approximately 15 nucleotides of nascent DNA. For many helicases, oligomeric state plays an important role in regulating enzymatic activity. Previous studies indicated that multiple ZRANB3 molecules can bind to a single forked DNA substrate; however, the functional role of ZRANB3 oligomerization remains unclear. Here, we employed single-molecule fluorescence techniques combining photobleaching and fluorescence resonance energy transfer to investigate the oligomeric behavior and kinetics of ZRANB3 during fork reversal and DNA unwinding. Both monomeric and dimeric ZRANB3 efficiently catalyze fork reversal, and the oligomeric state remains unchanged during the reaction. ZRANB3 removes RPA bound at the lagging-strand gap without reducing reaction efficiency, whereas RPA at the leading-strand gap inhibits fork reversal while leaving ZRANB3 oligomerization unaffected. At high protein concentrations, ZRANB3 is capable of unwinding nascent leading strand DNA up to 30 nucleotides in length. Concentration-dependent analyses show that DNA unwinding follows a sigmoidal dependence on ZRANB3 concentration, whereas RPA removal exhibits a hyperbolic trend. Although the proportion of dimeric ZRANB3 bound to DNA increases with protein concentration, no enhancement in unwinding rate or efficiency is observed for the dimer compared to the monomer. Overall, the sigmoidal behavior of DNA unwinding likely arises from changes in ZRANB3 oligomeric distribution and DNA binding dynamics rather than intrinsic catalytic differences between oligomeric states. This study provides single-molecule insights into the regulatory mechanisms of ZRANB3 in fork reversal and DNA unwinding.