Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg SE 41296, Sweden.
Institut Jacques Monod, Université de Paris, CNRS, UMR7592, Paris, France.
Nucleic Acids Res. 2021 Mar 18;49(5):2629-2641. doi: 10.1093/nar/gkab083.
We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.
我们使用单分子技术来描述非同源末端连接（NHEJ）修复原核 DNA 的动态过程，该系统仅由二聚体 Ku 和 Ligase D（LigD）组成。Ku 同源二聚体本身可以在钝末端 DNA 之间形成一个约 2 秒的联会，当加入 LigD 时，联会时间增加到约 18 秒，这一过程依赖于 Ku 的 C 端臂。对于 4 个 nt 的互补 DNA 突出端，联会寿命会急剧增加，而与 Ku 的 C 端臂无关。这些观察结果与不能桥接两个 DNA 底物的人类 Ku 形成鲜明对比。我们还证明，细菌 Ku 以协同的方式结合 DNA 末端以启动联会，并在连接完成后数小时内稳定地结合在 DNA 连接点上，这表明在体内存在一种用于去除蛋白质的系统。这些实验共同揭示了细菌 NHEJ 在 DNA 末端识别和处理中的动态过程。我们推测细菌和真核 NHEJ 之间存在进化上的相似性，并讨论了增加对细菌 NHEJ 的理解如何为针对该机制的未来抗生素治疗开辟道路。
