UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

UvrD 是非六聚体超家族 1 解旋酶的一种模型，它利用 ATP 水解沿着单链 DNA 逐步转位并解开双链体。以前对其步长的估计是间接的，对其步进机制缺乏共识。为了剖析 DNA 解旋的机制，我们使用光镊直接测量 UvrD 在处理 DNA 发夹时的步进行为，并表明 UvrD 表现出可变的步长，平均为 ~3 个碱基对。分析跨 ATP 的步进动力学揭示了以不同步长发生的催化事件的类型和数量。这些单分子数据揭示了一种机制，其中 UvrD 一次移动一个碱基对，但隔离新生的单链，在可变数量的催化循环后不均匀地释放它们。分子动力学模拟指出了这种行为的结构基础，确定了负责链螯合的蛋白质-DNA 相互作用。基于结构和序列比对数据，我们提出这种步进机制可能在其他非六聚体解旋酶中是保守的。