日本东京大学Hiroshi Nishimasu等研究人员合作揭示桥RNA引导重组的结构机制。2024年6月26日出版的《自然》杂志发表了这项成果。

研究人员最近发现，IS110家族元件编码一种重组酶和一种非编码桥RNA（bRNA），后者通过两个可编程环路赋予靶标DNA和供体DNA模块特异性。研究人员报告了IS110重组酶在重组反应周期的三个不同阶段与bRNA、靶标DNA和供体DNA复合物的冷冻电镜结构。IS110突触复合物由两个重组酶二聚体组成，其中一个包含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。研究人员发现RuvC-Tnp的活性位点横跨两个二聚体，催化丝氨酸残基位于靶标DNA和供体DNA的重组位点附近。

对这三种结构进行比较后发现：（1）靶标DNA和供体DNA的顶链在复合活性位点处被切割，形成共价的5′-磷酸丝氨酸中间体；（2）被切割的DNA链发生交换和聚合，形成霍利迪接合中间体；（3）该中间体随后通过切割底链而被分解。总之，这项研究揭示了双特异性RNA赋予IS110重组酶靶标DNA和供体DNA特异性以实现可编程DNA重组的机制。

研究人员表示，插入序列（IS）元件是原核生物基因组中最简单的自主转座元件。

附：英文原文

Title: Structural mechanism of bridge RNA-guided recombination

Author: Hiraizumi, Masahiro, Perry, Nicholas T., Durrant, Matthew G., Soma, Teppei, Nagahata, Naoto, Okazaki, Sae, Athukoralage, Januka S., Isayama, Yukari, Pai, James J., Pawluk, April, Konermann, Silvana, Yamashita, Keitaro, Hsu, Patrick D., Nishimasu, Hiroshi

Issue&Volume: 2024-06-26

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.

DOI: 10.1038/s41586-024-07570-2

Source: https://www.nature.com/articles/s41586-024-07570-2