Chen Xuemin, Yao Liangrui, Ma Shanshan, Yuan Xingyun, Yang Yang, Yuan Yuan, Liu Yumei, Liu Lan, Wang Huaibin, Yang Wei, Gellert Martin
Anhui university.
National Institutes of Health.
Res Sq. 2025 Feb 12:rs.3.rs-5443361. doi: 10.21203/rs.3.rs-5443361/v1.
The RAG1/2 recombinase, which initiates V(D)J recombination in jawed vertebrates, evolved from RNaseH-like transposases such as Transib and ProtoRAG . However, its post-cleavage transposase activity is strictly suppressed. Previous structural studies have focused only on the conserved core domains of RAG1/2, leaving the regulatory mechanisms of the non-core regions unclear. To investigate how RAG1/2 suppresses transposition and regulates DNA cleavage, we determined cryo-EM structures of nearly full-length RAG1/2 complexed with cleaved Recombination Signal Sequences (RSS) in a Signal-End Complex (SEC), at resolutions up to 2.95 Å. Two key structures, SEC-0 and SEC-PHD, reveal distinct regulatory roles of RAG2, which is absent in Transib transposase. SEC-0 displays a closed conformation, revealing that the core RAG2 facilitates sequential DNA cleavage by stabilizing the RSS-cleaved states in a "spring-loaded" mechanism. SEC-PHD reveals how RAG2's non-core PHD and Acidic Hinge (AH) domains, which are absent in ProtoRAG, inhibit target DNA binding in transposition. Histone H3K4me3, which recruits RAG1/2 to RSS sites, does not influence RAG1/2 binding to V, D or J gene segments bordered by RSS . In contrast, the suppressed transposition can be activated by H3K4me3 peptides that dislodge the inhibitory PHD domain . To achieve this de-repression in vivo, however, would require an unlikely close placement of two nucleosomes flanking a target DNA bent by nearly 180°. Our structural and biochemical results elucidate how RAG1 has acquired RAG2 and utilizes its core and non-core domains to enhance V(D)J recombination and suppress transposition.
RAG1/2重组酶在有颌脊椎动物中启动V(D)J重组，它由诸如Transib和ProtoRAG等类似RNaseH的转座酶进化而来。然而，其切割后的转座酶活性受到严格抑制。先前的结构研究仅聚焦于RAG1/2的保守核心结构域，非核心区域的调控机制尚不清楚。为了研究RAG1/2如何抑制转座并调节DNA切割，我们确定了近乎全长的RAG1/2与切割后的重组信号序列（RSS）在信号末端复合物（SEC）中复合的冷冻电镜结构，分辨率高达2.95 Å。两个关键结构，SEC-0和SEC-PHD，揭示了RAG2独特的调控作用，而Transib转座酶中不存在RAG2。SEC-0呈现出封闭构象，表明核心RAG2通过“弹簧加载”机制稳定RSS切割状态，促进DNA的顺序切割。SEC-PHD揭示了ProtoRAG中不存在的RAG2的非核心PHD和酸性铰链（AH）结构域如何在转座过程中抑制靶DNA结合。招募RAG1/2到RSS位点的组蛋白H3K4me3并不影响RAG1/2与由RSS界定的V、D或J基因片段的结合。相反，H3K4me3肽可激活被抑制的转座，这些肽会使抑制性的PHD结构域移位。然而，要在体内实现这种去抑制，需要两个核小体在一个弯曲近180°的靶DNA两侧紧密排列，这可能性不大。我们的结构和生化结果阐明了RAG1如何获得RAG2并利用其核心和非核心结构域来增强V(D)J重组并抑制转座。
