Recombination By-products Contribute to RAG-Mediated Chromosomal Alterations

Between 30% and 40% of lymphoid malignancies bear the hallmarks of errors in the recombination reactions that generate B- and T-cell receptor genes. Kirkham and colleagues identify a molecular mechanism that generates the substrates for end donation reactions, an erroneous V(D)J recombination reaction, where broken recombination signal sequences (RSS) become joined with independently generated double-strand breaks (DSB). They examined RAG interactions with the signal joint (SJ) in vitro and found that SJ–RSS complexes are cleaved asymmetrically. This raised the possibility that asymmetric cleavage events by complexes containing RAG and the excised genomic DNA [the excised signal circle (ESC)] could generate potential substrates for end donation reactions by causing double-strand break formation and leading to potential translocation events. They also characterized the stability of RAG:RSS post-cleavage complexes and observed that although the signal and coding ends of an RSS are released following SJ-stimulated cleavage, the RAG proteins remain stably associated with the SJ. This implies that the now-cut RSS is now a free substrate for joining with other DNA ends whereas the RAG complex can go on to trigger additional breaks at additional RSSs. The authors analyzed RAG–SJ cutting in cells transduced with either a 12-RSS or a 23-RSS along with either a wild-type or mutant SJ and found that the asymmetric SJ–RSS cutting reaction can be observed in vivo. Transfection of a RAG–ESC complex increased γH2AX foci, indicating that this free RAG complex can trigger genome-wide DSBs. Linear amplification–mediated high-throughput genome-wide translocation sequencing on SJ-transfected cells revealed a significant increase in translocation events involving broken RSSs. Intriguingly, the presence of the SJ increased the frequency of breaks in 8 to 10 of the 11 most commonly mutated genes in acute lymphoblastic leukemia (ALL). This recombination reaction by-product therefore serves as an unexpected potential underlying cause of lymphoid cell transformation.

Kirkham CM, Scott JNF, Wang X, Smith AL, Kupinski AP, Ford AM, et al. Cut-and-run: A distinct mechanism by which V(D)J recombination causes genome instability. Mol Cell 2019 Mar 18 [Epub ahead of print].

Note: Research Watch is written by Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details. For more Research Watch, visit Cancer Discovery online at http://cancerdiscovery.aacrjournals.org/CDNews.