RAG endonuclease initiates V(D)J recombination by binding to the recombination signal sequences (RSSs), introducing DNA breaks between two V(D)J gene segments, and mediating their joining. This process is tightly regulated to generate diverse antigen receptor repertoires and prevent aberrant events that could cause genomic translocations/deletions and lymphoid cancer. RAG can track directionally over chromosomal loop domain in which they reside. Tracking is evidenced by cleavage/joining of short cryptic RSSs lying exclusively in convergent orientation to a bona fide RSS. However, the mechanism of RAG tracking and its impacts remain unknown. Here I established an unbiased assay system by utilizing allele-specific barcode and employing high throughput genome-wide translocation sequencing (HTGTS) to quantitatively evaluate RAG-mediated recombination frequency. I found that insertion of one single bona fide RSS is sufficient to initiate RAG tracking in the c-Myc domain and the recombination between the RSS and its cryptic RSSs seems to follow the 12/23 rule to some degree. When ectopically inserted bona fide RSS pair is 0.5kb apart, convergent configuration of the RSS pair is 4.8-fold more frequent than the tandem configuration. When the distance increases to 267kb, the difference increases to 14-fold, suggesting the contribution of RAG tracking over diffusion-based mechanism increases when the distance between RSS pair increases. In addition, the presence of CTCF-binding element promotes recombination by more than 10-fold. Depletion of Wapl, cohesin remover, to disrupt cohesin turnover reduces V(D)J recombination. The distal RSSs are much preferentially affected. The data suggest that RAG tracking may be driven by cohesin sliding along chromatin.

Citation Format: Cheng-Sheng Lee, Jiazhi Hu, Frederick W. Alt. Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B171.