V(D)J Recombination Signal Sequence Biases Antigen Receptor Repertoire and Promotes Off-Target Activity 3164

Description V(D)J recombination is performed by the recombination activating genes 1 and 2 (RAG1/2) enzyme complex, which leads to antigen receptor (AgR) repertoires in immune systems. To initiate this process, RAG1/2 cleaves at recombination signal sequences (RSSs) located within AgR loci. RSSs in AgR loci vary substantially in sequence from a canonical RSS and yet still act as substrates for recombination. Further, RSS-like elements in other areas of the genome can lead to inappropriate recombination, potentially leading to certain forms of leukemia and lymphoma. We hypothesize that the efficiency of recombination is governed by intrinsic properties embedded in the DNA sequence. To identify the basis for RSS utilization in V(D)J recombination, a novel next generation sequencing (NGS) method was used to interrogate how recombination efficiency was affected by varying nucleotide substitutions. In addition, variant RSSs were tested by biochemical studies to identify the basis of impaired recombination. Results indicate the extent that sequence variations in the RSS can affect specific steps of the recombination reaction. Despite deviating from the canonical RSS, we propose that sufficient intrinsic properties of certain variant RSSs are maintained to allow V(D)J recombination activity. These results can provide improved predictions of recombination efficiency at antigen receptor genes, as well as identify RSS-like sequences that result in potential genomic translocations and oncogenesis. Funding Sources NIH 1R01AI168175; OCAST HR21-142 Topic Categories Hematopoiesis and Immune System Development (HEM)