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Intermediates in V(D)J recombination: a stable RAG1/2 complex sequesters cleaved RSS ends.

J M Jones1, M Gellert

  • 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 241, Bethesda, MD 20892, USA.

Proceedings of the National Academy of Sciences of the United States of America
|October 19, 2001
PubMed
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The RAG1/2 recombinase enzyme forms stable complexes with cleaved DNA signal ends after gene rearrangement. These complexes prevent DNA joining and cell cycle checkpoint activation, explaining the persistence of signal ends.

Area of Science:

  • Molecular Biology
  • Immunology
  • Genetics

Background:

  • Antigen receptor gene rearrangement is crucial for adaptive immunity.
  • The RAG1/2 recombinase enzyme mediates this V(D)J recombination process.
  • Understanding RAG1/2 function is key to immune system development and function.

Purpose of the Study:

  • To investigate the fate of DNA signal ends after RAG1/2-mediated cleavage.
  • To determine the stability and characteristics of RAG1/2-bound signal end complexes.
  • To elucidate the mechanism preventing DNA joining and cell cycle activation.

Main Methods:

  • Biochemical analysis of RAG1/2-DNA complexes.
  • Heparin treatment to assess complex stability.
  • In vitro DNA joining assays using purified mammalian joining apparatus proteins (Ku70/86, XRCC4, DNA ligase IV).

Related Experiment Videos

Main Results:

  • Cleaved DNA signal ends remain stably associated with RAG1/2 in heparin-resistant complexes.
  • These signal end complexes sequester the DNA ends, preventing access by the joining apparatus.
  • Deproteinized cleaved ends can be joined, but RAG1/2 complexation inhibits this process.

Conclusions:

  • RAG1/2-mediated cleavage results in stable, sequestered signal end complexes.
  • This sequestration mechanism explains the persistence of signal ends and lack of DNA damage response.
  • The findings provide insight into the regulation of V(D)J recombination and genome stability.