Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes¹. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops². Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC–Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5′-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.

插入序列（IS）元件是原核基因组中发现的最简单的自主转座元件¹ 。我们最近发现 IS110 家族元件编码重组酶和非编码桥 RNA (bRNA)，通过两个可编程环赋予靶 DNA 和供体 DNA 模块化特异性² 。在此，我们报告了 IS110 重组酶与其 bRNA、靶 DNA 和供体 DNA 在重组反应周期的三个不同阶段的复合物的冷冻电子显微镜结构。 IS110突触复合体包含两个重组酶二聚体，其中一个容纳bRNA的靶结合环并与靶DNA结合，而另一个则协调bRNA供体结合环和供体DNA。我们发现了跨越两个二聚体的复合 RuvC-Tnp 活性位点的形成，将催化丝氨酸残基定位在靶 DNA 和供体 DNA 中的重组位点附近。三种结构的比较表明，(1) 靶标和供体 DNA 的顶链在复合活性位点处被切割，形成共价 5'-磷酸丝氨酸中间体，(2) 切割的 DNA 链被交换并重新连接以产生 Holliday连接中间体，并且(3)该中间体随后通过底部链的切割而被解析。总体而言，这项研究揭示了双特异性 RNA 赋予 IS110 重组酶靶标和供体 DNA 特异性以实现可编程 DNA 重组的机制。