RAG-1 and RAG-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution

Area of Science:

• Molecular Biology
• Immunology
• Genetics

Background:

• V(D)J recombination is essential for adaptive immunity, involving RAG-1 and RAG-2 proteins.
• RAG proteins introduce DNA breaks at recombination signal sequences (RSSs).
• The precise roles of RAG-1 and RAG-2 in initial DNA recognition remain debated.

Purpose of the Study:

• To develop an assay for assessing functional RAG-RSS complex formation and stability.
• To elucidate the roles of RAG-1 and RAG-2 in complex formation prior to DNA cleavage.
• To investigate how RSS mutations affect RAG-substrate complex stability and cleavage.

Main Methods:

• Developed an in vitro assay to monitor RAG-RSS complex formation and DNA cleavage.
• Utilized substrate mutations within the RSS nonamer and heptamer.
• Assessed complex stability through a sequestration assay under in vitro cleavage conditions.

Main Results:

• RAG proteins form a stable complex with intact DNA before strand scission.
• Both RAG-1 and RAG-2 are required for stable complex formation.
• Nonamer mutations destabilize the RAG-substrate complex, while heptamer mutations primarily affect hairpin formation without significantly impacting complex stability.

Conclusions:

• The RAG-RSS complex is pre-formed and stable prior to DNA cleavage.
• RAG-1 and RAG-2 cooperatively bind substrate DNA, essential for complex stability.
• RSS sequence elements differentially impact RAG complex stability and cleavage outcomes.