ABSTRACT
Antigen receptor (AgR) diversity is central to the ability of adaptive immunity in jawed vertebrates to protect against pathogenic agents. The production of highly diverse AgR repertoires is initiated during B and T cell lymphopoiesis by V(D)J recombination, which assembles the receptor genes from component gene segments in a cut-and-paste recombination reaction. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank AgR gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence, and the molecular mechanism for semiselective V(D)J recombination specificity is unknown. The modulation of chromatin structure during V(D)J recombination is essential in the formation of diverse AgRs in adaptive immunity while also reducing the likelihood for off-target recombination events that can result in chromosomal aberrations and genomic instability. Here we review what is presently known regarding mechanisms that facilitate assembly of RAG1/2 with RSSs, the ensuing conformational changes required for DNA cleavage activity, and how the readout of the RSS sequence affects reaction efficiency.
ABSTRACT
In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.
