Sun Bo, Wei Kong-Ji, Zhang Bo, Zhang Xing-Hua, Dou Shuo-Xing, Li Ming, Xi Xu Guang
Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
EMBO J. 2008 Dec 17;27(24):3279-87. doi: 10.1038/emboj.2008.240. Epub 2008 Nov 13.
Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3'-5' polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (approximately 40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from approximately 40 to approximately 150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from approximately 45 to approximately 10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.
大肠杆菌UvrD是一种非环状的解旋酶模型，在DNA解旋过程中表现出3'-5'极性。我们使用横向磁镊和DNA发夹，测量了UvrD在各种使DNA不稳定的力作用下的解旋动力学。解旋爆发的多种形式和停顿时间的分布支持UvrD以二聚体形式解旋DNA的机制。二聚体的两个亚基协同作用，持续地解旋DNA。它们可以共同切换链，并在另一条链上向后移位以实现缓慢（约40 bp/s）的重新缠绕，或者同时解离以实现快速重新杂交。二聚体的部分解离会导致解旋过程中间出现停顿，或者在重新缠绕过程中间将移位速率从约40 nt/s提高到约150 nt/s。此外，令人惊讶的是，当力从2 pN增加到12 pN时，解旋速率从约45 bp/s降低到约10 bp/s。这些结果导致了一种应变尺蠖机制，其中需要一种使单链DNA弯曲和拉紧的构象变化来激活二聚体。
