RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of ‘alternative NHEJ’

DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone pro-cess termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physio-logic DSBs remains unknown. Here, we show that deletion of RAG2’s C-terminus allows a-NHEJ to re-pair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase in-fluences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expecta-tion, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appre-ciated. Whole genome sequencing of a lymphoma arising in a p53−/ − mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites