“Cut-and-Run”: A New Way By Which Aberrant V(D)J Recombination Causes Genome Instability

V(D)J recombination is essential to create a highly diverse repertoire of antigen receptors. However, this process carries an inherent risk because it involves breaking genomic DNA. Indeed, errors in this process are a major
cause of leukaemias and lymphomas. Previous studies showed that the recombination by-product, the excised signal circle (ESC), can be a source of genome instability. The Boyes lab showed recently that the ESC triggers cutting at a recombination signal sequence (RSS) but the ESC itself is largely uncut i.e. asymmetric cleavage occurs in an ESC/RSS synaptic complex. Since the ESC remains bound by recombinase (RAG) proteins, this suggests that the ESC can trigger cutting at one RSS and then “run” to trigger cutting at
further RSS or cryptic RSS (cRSS) in a reaction that was named “cut-and-run”. This further implies that the ESC could trigger numerous double strand breaks (DSBs) in the genome, leading to genome instability.
I further investigated the mechanism of cut-and-run.Firstly, I examined the reaction in vitro and showed that the asymmetric cleavage is due to RAGs simultaneously binding to both RSSs in the ESC and blocking cutting at heptamer/heptamer boundary of ESC. Next, I showed this asymmetric cutting also occurs in vivo. Moreover, if I mutate one of the RSSs in the ESC, both ESC and RSS are cut.
Next, I investigated the dangers of cut-and-run. Firstly, I showed that it generates one blunt signal end and one hairpinned coding end that are not readily repaired. Secondly, ESC was found to trigger cutting at cRSS in vitro.
Thirdly, I showed that this reaction can cause DSBs throughout the genome. Lastly, the lambda and kappa ESCs were found to persist to mature B cells.
Together, these investigations suggest that cut-and-run is a dangerous reaction by providing substrates that potentially lead to chromosome instabilities.