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Nat Struct Mol Biol. 2018 Aug;25(8):732-742. doi: 10.1038/s41594-018-0098-5. Epub 2018 Jul 30.

DNA melting initiates the RAG catalytic pathway.

Ru H1,2, Mi W3, Zhang P1,2, Alt FW2,4,5, Schatz DG6, Liao M7, Wu H8,9.

Author information

1
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
2
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
3
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
4
Department of Genetics, Harvard Medical School, Boston, MA, USA.
5
Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.
6
Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.
7
Department of Cell Biology, Harvard Medical School, Boston, MA, USA. maofu_liao@hms.harvard.edu.
8
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. wu@crystal.harvard.edu.
9
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA. wu@crystal.harvard.edu.

Abstract

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.

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