DnaB Helicase Activity Is Modulated by DNA Geometry and Force
DnaB Helicase Activity Is Modulated by DNA Geometry and Force
复制标题
DOI：
10.1016/j.bpj.2010.07.039
复制
发表时间：
2010-10-06
影响因子：
3.4
通讯作者：
Saleh, Omar A.
中科院分区：
文献类型：
--
作者：
Ribeck, Noah;Kaplan, Daniel L.;Saleh, Omar A.
大肠杆菌的复制解旋酶是 DnaB，这是一种六聚体环形运动蛋白，可环绕 ssDNA 并沿 ssDNA 易位，在运动前解开 dsDNA。 DnaB 的微观机制尚不清楚；此外，之前的工作发现 DnaB 的活性会被其他复制蛋白修饰，表明其具有一定的机制灵活性。为了研究这些问题，我们量化了三个受张力束缚的 DNA 几何结构中单个 DnaB 分子的易位和解旋。我们的数据支持以下结论：1) DnaB 的解旋因力诱导的 dsDNA 不稳定而增强。 2)，这种刺激的强度随着施加到 DNA 底物上的张力的几何形状而变化，可能是由于解旋酶和封闭的 ssDNA 链之间的相互作用。 3)、DnaB 解旋和（在较小程度上）易位被暂停打断，这也取决于力和 DNA 几何形状。 4)，当对解旋酶结合的链施加大的张力时，DnaB 移动得更慢，这表明它必须执行机械功来抵抗所施加的力压紧链。我们的结果对 DnaB 易位和解旋的分子机制以及调节 DnaB 活性的方法具有影响。
The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.