1] Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain [2] CIBER-BBN de Bioingenieria, Biomateriales y Nanomedicina, Instituto de Sanidad Carlos III, Madrid, Spain.
Nat Commun. 2013;4:2368. doi: 10.1038/ncomms3368.
Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.
在 DNA 修复和基因重组中，既能解开又能重新缠绕 DNA 的解旋酶起着核心作用。与解开相比，由于其热力学上的下坡性质，解旋酶介导的 DNA 重新缠绕在生化特性上很难被描述。在这里，我们使用单分子测定法来机械地破坏 DNA 分子，并实时跟踪两种 DNA 修复解旋酶，噬菌体 T4 UvsW 和大肠杆菌 RecG 的解开和重新缠绕。我们发现两种酶都是强大的重新缠绕酶，可以抵抗高达 35 pN 的反向力，揭示了它们的活性特征。在重新缠绕反应过程中产生的功使它们能够将重新缠绕与 DNA 解开和/或蛋白质位移反应耦合，这对停滞的 DNA 复制叉的挽救至关重要。整体结果支持单体重新缠绕酶的一般机制。
