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Cracking the DNA Code for V(D)J Recombination.

Min-Sung Kim1, Watchalee Chuenchor2, Xuemin Chen2

  • 1Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892, USA; Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Republic of Korea.

Molecular Cell
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Summary

The RAG1/2 recombinase enzyme uses DNA bending and structural changes to initiate V(D)J recombination, a critical process for adaptive immunity in vertebrates. These structural insights explain DNA pairing rules and hairpin formation during this immune system development.

Keywords:
CA/TG dinucleotidesDNA bendingDNA hairpinNBDSCIDpolypurine tract

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Area of Science:

  • Structural Biology
  • Immunology
  • Molecular Biology

Background:

  • V(D)J recombination is essential for adaptive immunity in vertebrates.
  • The RAG1/2 recombinase enzyme initiates this process by cleaving DNA at specific recombination signal sequences (RSS).
  • Understanding the structural mechanisms of RAG1/2 is key to comprehending immune system development.

Purpose of the Study:

  • To determine the high-resolution structures of the RAG1/2 recombinase with DNA during key steps of V(D)J recombination.
  • To elucidate the dynamic conformational changes in both protein and DNA during the reaction.
  • To understand the structural basis for DNA bending, pairing, and hairpin formation.

Main Methods:

  • X-ray crystallography and cryo-electron microscopy (cryo-EM) were used to obtain structures.
  • Structures were determined for pre-reaction and hairpin-forming complexes.
  • High-resolution imaging up to 2.75 Å was achieved.

Main Results:

  • RAG1/2 and DNA exhibit significant structural plasticity and undergo dramatic conformational changes.
  • DNA bending of 60° and 150° in 12- and 23-RSS spacers is crucial for RAG1/2 function.
  • Two RAG1 subunits interact extensively with the bent 12/23-RSS DNA substrates.
  • Catalysis in crystallo revealed DNA hairpin formation and stabilization via base stacking.

Conclusions:

  • DNA bending and structural plasticity are fundamental to RAG1/2 function in V(D)J recombination.
  • The observed DNA pairing is sequence-context and structure-specific, explaining "beyond 12/23" restrictions.
  • The structures provide unprecedented insight into the mechanism of DNA hairpin formation and stabilization.