V(D)J recombination assembles functional antigen receptor genes from component gene segments to produce the diverse repertoire of functional immunoglobulin and T cell receptors in B and T lymphocytes, respectively. RAG1 and RAG2 are lymphoid-specific proteins that catalyze the DNA cleavage steps in V(D)J recombination. RAG-mediated DNA cleavage activity is directed to discrete DNA sequences known as recombination signal sequences (RSSs) that flank the coding gene segments in the antigen receptor loci. In individual recombination reactions, a heterotetrameric RAG1/2 complex binds simultaneously to two RSSs and creates DNA double strand breaks at the border between each RSS and the adjoining coding segment. Joining of the coding segments is carried out by ubiquitous DNA repair factors. Many RSSs are only semi-conserved, such that recombination of poorly conserved RSSs requires promiscuous RAG1/2 activity. RAG1/2 also creates aberrant recombination events at RSS-like sites, called cryptic RSSs (cRSS), located outside of the antigen receptor loci, which can cause oncogenic chromosomal rearrangements. Therefore, RAG1/2 must be promiscuous to facilitate recombination of poorly conserved RSSs, but it must also be precise to avoid off-target cRSSs. To characterize the DNA sequence specificity of RAG1/2, we are developing a high-throughput plasmid recombination method to analyze V(D)J recombination sequence specificity. Greater than 105 extrachromosomal V(D)J recombination substrates of differing sequences are transfected into RAG1/2 expressing cells, the resulting recombination products selectively amplified, and subsequently analyzed by next-generation sequencing. Using this method, we will empirically characterize RSS motifs that enhance RAG1/2 activity to shape a diverse antigen receptor repertoire, as well as identify suboptimal RSS motifs that favor nonconventional V(D)J recombination reactions. To date, highly informative results have been obtained from preliminary studies using this method, which suggest sequence interdependencies exist between different regions of the RSS with significant consequences on the level of V(D)J recombination activity. Furthermore, specific RSS motifs appear to preferentially favor nonconventional V(D)J recombination reactions. Based on our preliminary results, we hypothesize that specific interrelationships within RSSs 1) influence their relative utilization by the RAG proteins and 2) govern their fate in conventional versus aberrant V(D)J recombination reactions.
Our aims are to analyze separate regions within the RSS for their effect on V(D)J recombination activity, and second, identify RSS motifs that skew the V(D)J recombination reaction to the formation of aberrant products. Overall, we predict that findings from this project will significantly improve our current understanding of RAG selectivity of RSSs and cRSSs in normal and aberrant V(D)J recombination reactions, respectively.
In developing B and T lymphocytes, functional antigen receptor genes are assembled from component gene segments by V(D)J recombination through a DNA cleavage and joining mechanism. In this project, we will develop a cellular recombination assay coupled with a high throughput sequencing approach to decipher patterns in DNA sequences that govern the efficacy of V(D)J recombination. Findings from this study will be important for elucidating how the antigen receptor repertoire in the adaptive immune system is formed, as well as the basis for aberrant recombination reactions that can lead to oncogenic chromosomal rearrangements.