PDF(Pubmed)
Abstract：
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.
摘要：
插入序列（IS）元件是在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),其通过两个可编程环2赋予靶DNA和供体DNA的模块特异性。在这里，我们报道了IS110重组酶与其bRNA复合的低温电子显微镜结构，目标DNA和供体DNA在重组反应循环的三个不同阶段。IS110突触复合物包含两个重组酶二聚体，其中一个包含bRNA的靶结合环并与靶DNA结合，而另一个协调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和供体DNA中的重组位点附近。三种结构的比较表明：（1）靶和供体DNA的顶部链在复合活性位点被切割，形成共价5'-磷酸丝氨酸中间体，（2）切割的DNA链交换和重新连接，以创建霍利迪连接中间体，和(3)该中间体随后通过底部链的裂解而分解。总的来说,这项研究揭示了双特异性RNA赋予IS110重组酶靶和供体DNA特异性以进行可编程DNA重组的机制。
