Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
EMBO J. 2021 Jan 4;40(1):e105666. doi: 10.15252/embj.2020105666. Epub 2020 Oct 2.
Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
复制-粘贴转位是一种主要的细菌 DNA 迁移途径。它通过在整合后上调下游基因的表达，显著促进了抗生素耐药性的出现。与其他转位途径不同，它需要非对称和对称链转移步骤。在这里，我们报告了第一个复制-粘贴转座酶的结构研究，并证明了它在体外催化所有途径步骤的能力。IS256 家族插入序列的 ISCth4 转座酶的 X 射线结构，与反应中的三个连续步骤对应的 DNA 底物结合，揭示了一种不寻常的非对称二聚体转座体。在转位过程中，一系列 N 端结构域结合单个转座子末端，而催化结构域移动以适应不同的底物。这些构象变化控制了转座子末端侧翼 DNA 的路径，并产生了 DNA 结合位点。我们的结果解释了初始链转移的非对称结果，并展示了 DNA 结合如何通过不对称转座酶进行调节，从而允许捕获第二个转座子末端，并整合一个环状中间体。
