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Related Experiment Videos

Ordered DNA release and target capture in RAG transposition.

Adam G W Matthews1, Sheryl K Elkin, Marjorie A Oettinger

  • 1Department of Molecular Biology, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA.

The EMBO Journal
|February 28, 2004
PubMed
Summary
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The RAG1/2 enzyme

Area of Science:

  • Molecular Biology
  • Immunology
  • Genetics

Background:

  • V(D)J recombination is a crucial process in adaptive immunity, generating diverse antibody and T-cell receptor genes.
  • The RAG1/2 complex mediates V(D)J recombination through DNA cleavage and subsequent joining or transposition.
  • Understanding the precise mechanisms of RAG1/2 action, including transposition, is key to comprehending immune system development.

Purpose of the Study:

  • To elucidate the sequential steps and molecular requirements of RAG-mediated transposition.
  • To investigate the role of the RAG1/2 active site, specifically the DDE motif, in target DNA capture during transposition.
  • To determine the fate of coding DNA flanking the V(D)J cleavage site during transposition.

Main Methods:

  • Biochemical assays to monitor DNA cleavage, release, and target capture by the RAG1/2 complex.

Related Experiment Videos

  • Analysis of RAG1/2 active site mutants, focusing on the DDE motif, to assess their function in transposition.
  • Detection of DNA species within the target capture complex.

    • Main Results:

    • V(D)J cleavage and release of flanking coding DNA precede target DNA capture in transposition.
    • No coding DNA is detected in the target capture complex.
    • The RAG1/2 DDE motif is essential for target DNA capture, distinct from its role in cleavage.

    Conclusions:

    • The RAG1/2 transposase likely possesses a single binding site for non-RSS DNA, accommodating either target or coding DNA, but not both.
    • The sequential requirement for coding DNA release before target capture suggests a mechanism to prevent aberrant transpositional resolution of V(D)J recombination intermediates.
    • This finding clarifies the mechanistic constraints on RAG1/2 activity, ensuring proper immune gene formation.