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Related Experiment Videos

Assembly of the RAG1/RAG2 synaptic complex.

Cynthia L Mundy1, Nadja Patenge, Adam G W Matthews

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

Molecular and Cellular Biology
|December 12, 2001
PubMed
Summary
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The RAG1/RAG2 recombinase complex assembles on a single DNA signal sequence, but requires a second complementary signal to initiate V(D)J recombination and DNA cleavage. This binding order may dictate preferred assembly sites in vivo.

Area of Science:

  • Molecular Biology
  • Immunology
  • Genetics

Background:

  • V(D)J recombination is crucial for adaptive immunity, assembling antigen receptor genes.
  • This process relies on the RAG1/RAG2 recombinase recognizing specific DNA signal sequences.
  • Cleavage by RAG1/RAG2 is restricted and requires the formation of a synaptic complex between two signal elements.

Purpose of the Study:

  • To investigate the initial binding and assembly of RAG proteins on signal sequences.
  • To understand the requirements for RAG protein complex activation and DNA cleavage.
  • To determine if a preferred site for RAG protein assembly exists during V(D)J recombination.

Main Methods:

  • Analysis of RAG protein binding to single and paired DNA signal sequences.

Related Experiment Videos

  • Biochemical assays to assess complex formation and DNA cleavage activity.
  • Investigating the stoichiometry of RAG1 and RAG2 proteins in the initial complex.

    • Main Results:

    • The RAG1/RAG2 complex can assemble on a single DNA signal sequence.
    • This initial complex, comprising RAG2 dimers and RAG1 trimers, is inactive for DNA cleavage.
    • A second, complementary signal sequence is necessary to activate the complex and induce double-strand break formation.
    • This activation may involve a conformational change induced by the second signal's binding.

    Conclusions:

    • RAG protein assembly on DNA signals is a stepwise process.
    • The binding of the second signal sequence triggers the catalytic activity of the RAG complex.
    • In vivo, one signal sequence might serve as a preferential site for the initial assembly of the RAG1/RAG2 complex during V(D)J recombination.