Department of Molecular Biology, Graduate School of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588 Japan.
Commun Biol. 2019 Nov 14;2:413. doi: 10.1038/s42003-019-0655-4. eCollection 2019.
Bacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.
细菌 RecN 与结构维持染色体 (SMC) 蛋白家族密切相关，通过同源重组在 DNA 双链断裂 (DSB) 的修复中发挥作用。在这里，我们表明纯化的 RecN 蛋白在拓扑结构上加载到单链 DNA (ssDNA) 和双链 DNA (dsDNA) 上，ssDNA 具有优先性。RecN 在拓扑结构上结合的 dsDNA 会从线性 dsDNA 的末端滑出，但 ssDNA 上的 RecA 核蛋白丝会阻止这种情况发生，从而允许 RecN 转移到 DSB 上。此外，我们发现，一旦 RecN 被招募到 ssDNA 上，它就可以以 ATP 依赖性的方式在拓扑结构上捕获第二个 dsDNA 底物，这表明它在联会中发挥作用。事实上，RecN 刺激 RecA 介导的 D 环形成和随后的链交换活性。我们的发现为 RecN 被招募到 DSB 以及 RecN 介导的姐妹染色单体相互作用提供了机制上的见解，这两者都在 RecA 介导的 DSB 修复中发挥作用。
