利用單分子螢光方法研究 RPA 調控複製差反轉酵素SMARCAL1的作用機制

Abstract

DNA 複製叉前方的 DNA 損傷會導致 DNA 複製停滯，並造成基因體不穩定。為了維持複製叉的穩定並啟動後續的 DNA 修復路徑，複製叉重塑酵素會將停滯的複製叉處理為反轉的複製叉。在人類中，SMARCAL1 被認為是參與複製叉反轉早期階段的重要蛋白之一，並受到replication protein A (RPA) 的調控。RPA 是一種高親和力的單股DNA結合蛋白，在複製叉停滯時會優先結合於暴露的單股上。先前研究指出，RPA 可促進 SMARCAL1 在前進股缺口複製叉上的活性，同時抑制其在延遲股缺口複製叉上的活性，然而其詳細的調控機制仍未明。為探討上述問題，本研究利用單分子螢光技術，建立複製叉反轉、解旋及黏合實驗並使用具螢光標記之 SMARCAL1 與 RPA進行觀測複製叉反轉。結果顯示，RPA 僅影響複製叉反轉的起始步驟，對後續四股結構的遷移並無顯著影響。螢光標記的 SMARCAL1 顯示其在複製叉DNA上具高度的動態結合和解離行為，動力學分析指出 RPA存在時，SMARCAL1 在前進股缺口為活化模型。依據反轉活性，可分為活性與非活性結合態；解旋實驗發現 SMARCAL1 具有新生股解旋活性，且該活性受到 RPA 調控，且螢光 SMARCAL1 訊號顯示，活性結合態可能與新生股解旋行為相關。而黏合實驗結果顯示，在前進股與延遲股缺口複製叉上 RPA 皆未對 SMARCAL1 誘導之 DNA 黏合反應造成抑制。綜合上述結果，本研究提出 RPA 調控 SMARCAL1 活性的可能機制，指出 RPA主要透過調控 SMARCAL1 結合於 DNA 的活性與非活性狀態的比例，進而影響其功能。

DNA lesions ahead of replication forks can stall DNA replication. To maintain DNA fork stability and activate downstream DNA repair pathways, fork-remodeling enzymes process stalled forks into reversed forks. In humans, SMARCAL1 is thought to function at the early stage of fork reversal and is regulated by replication protein A (RPA), a high-affinity single-stranded DNA-binding protein that binds exposed ssDNA upon fork stalling. Previous studies showed that RPA enhances SMARCAL1 activity on leading-gap forks while inhibiting it on lagging-gap forks; however, the underlying mechanism remains unclear. To investigate this regulatory mechanism, we developed single-molecule fluorescence assays to directly monitor fork reversal, unwinding, and annealing using fluorescently labeled SMARCAL1 and RPA. We show that, on leading-gap forks, RPA specifically affects the initiation of fork reversal but does not influence subsequent four-way junction migration. Fluorescent SMARCAL1 exhibits highly dynamic association and dissociation on fork DNA, and kinetic analysis indicates SMARCAL1 adapts activation model on RPA-coated leadin gap fork . We define SMARCAL1 binding into productive and non-productive states. In unwinding assays, SMARCAL1 exhibits short-distance nascent strand unwinding activity that is similarly regulated by RPA. The fluorescent SMARCAL1 signal suggests that the productive binding state correlates with nascent strand unwinding. In contrast, annealing assays show that RPA does not significantly inhibit SMARCAL1-mediated DNA annealing on either leading- or lagging-gap forks. Finally, we show that SMARCAL1 does not exhibit parental strand unwinding activity. Together, our results suggest that RPA regulates SMARCAL1 by modulating the distribution of productive and non-productive SMARCAL1 binding states, which is associated with nascent strand unwinding activity.