Summary: Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2. : DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. Off-target RAG cleavage has only been analyzed in the context of DNA repair defects. Here, Hewitt et al. identify a phosphorylation site on RAG2 that controls RAG cleavage to maintain genome stability independent of a repair defect. Keywords: RAG cleavage regulation, genome stability, V(D)J recombination, ATM, RAG2S365, reciprocal translocations, developing lymphocytes
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