V(D)J recombination and RAG-mediated transposition in yeast

Area of Science:

• Molecular Biology
• Immunology
• Genetics

Background:

• Vertebrate immune cells assemble antigen receptor genes via V(D)J recombination.
• This process utilizes lymphoid-specific RAG1 and RAG2 proteins.
• V(D)J recombination is typically confined to the vertebrate immune system.

Purpose of the Study:

• To investigate if murine RAG1 and RAG2 can function in a lower eukaryote.
• To determine if V(D)J recombination machinery can be reconstituted in Saccharomyces cerevisiae.
• To explore the mechanism of RAG-mediated DNA cleavage and joining in yeast.

Main Methods:

• Expression of murine RAG1 and RAG2 in Saccharomyces cerevisiae.
• Introduction of V(D)J recombination substrates into yeast cells.
• Analysis of DNA cleavage, signal end formation, and signal joint generation.
• Assessment of the role of yeast nonhomologous end-joining protein LIF1.

Main Results:

• RAG1 and RAG2 expression in yeast induced V(D)J cleavage and rejoining.
• Precise signal joints were formed, dependent on the yeast XRCC4 homolog LIF1.
• RAG proteins influenced signal-end joining, which is generally imprecise for blunt ends in this yeast strain.
• RAG-induced DNA cleavage also led to transposition of cleaved ends into new genomic sites.

Conclusions:

• The RAG proteins possess the intrinsic enzymatic activity to perform V(D)J recombination in yeast.
• Yeast nonhomologous end-joining pathways, specifically LIF1, participate in RAG-mediated DNA repair.
• Saccharomyces cerevisiae serves as a viable system for studying RAG protein function and V(D)J recombination in vivo.
• RAG-mediated transposition can be studied in yeast, offering new avenues for research.