Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
Nat Struct Mol Biol. 2018 Aug;25(8):732-742. doi: 10.1038/s41594-018-0098-5. Epub 2018 Jul 30.
The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of zebrafish RAG in complex with one or two intact recombination signal sequences (RSSs), at up to 3.9-Å resolution. Unexpectedly, these structures reveal DNA melting at the heptamer of the RSSs, thus resulting in a corkscrew-like rotation of coding-flank DNA and the positioning of the scissile phosphate in the active site. Substrate binding is associated with dimer opening and a piston-like movement in RAG1, first outward to accommodate unmelted DNA and then inward to wedge melted DNA. These precleavage complexes show limited base-specific contacts of RAG at the conserved terminal CAC/GTG sequence of the heptamer, thus suggesting conservation based on a propensity to unwind. CA and TG overwhelmingly dominate terminal sequences in transposons and retrotransposons, thereby implicating a universal mechanism for DNA melting during the initiation of retroviral integration and DNA transposition.
DDE 家族酶（包括 RAG 内切核酸酶）启动 DNA 切割的机制尚不清楚，该酶参与 V(D)J 重组的起始。本文报道了 6 种斑马鱼 RAG 与一个或两个完整重组信号序列（RSS）复合物的冷冻电镜结构，分辨率高达 3.9Å。出乎意料的是，这些结构显示 RSS 七聚体处的 DNA 熔解，导致编码侧翼 DNA 的螺旋式旋转，并使切割磷酸位于活性位点。底物结合与二聚体打开和 RAG1 中的活塞式运动相关，首先向外以容纳未熔解的 DNA，然后向内将熔解的 DNA楔入。这些预切割复合物显示 RAG 在七聚体保守末端 CAC/GTG 序列上的碱基特异性接触有限，这表明基于解旋倾向的保守性。CA 和 TG 在转座子和逆转座子的末端序列中占绝对优势，这表明在逆转录病毒整合和 DNA 转座起始过程中存在普遍的 DNA 熔解机制。
