Ha Taekjip, Rasnik Ivan, Cheng Wei, Babcock Hazen P, Gauss George H, Lohman Timothy M, Chu Steven
Department of Physics, University of Illinois, Urbana, Illinois 61801, USA.
Nature. 2002 Oct 10;419(6907):638-41. doi: 10.1038/nature01083.
Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.
解旋酶是一种马达蛋白，它将由ATP结合和水解诱导的构象变化与双链核酸的解旋偶联起来，并与多种人类疾病相关。一些解旋酶以六聚体环的形式发挥作用，但非六聚体解旋酶的功能形式一直存在争议。在这里，我们结合使用表面固定方案和单分子荧光测定法（这些方法不会干扰生物活性）来探测大肠杆菌Rep解旋酶对DNA的解旋作用。我们的研究表明，Rep单体利用ATP水解向单链和双链DNA的交界处移动，但随后会表现出不会导致DNA解旋的构象波动。只有通过额外的蛋白质结合形成功能性解旋酶时，DNA解旋才会启动。在解旋过程中功能性复合物的部分解离会导致中断（“停顿”），这要么导致复合物完全解离时双链重新缠绕，要么导致功能性解旋酶重新形成时解旋重新启动。这些结果表明，体外观察到的Rep解旋持续性较低是由于功能性复合物相对不稳定所致。我们预计这些技术将有助于对其他解旋酶和蛋白质-DNA相互作用进行动态研究。
