Cut-and-run: a new mechanism by which V(D)J recombination could trigger leukaemia and lymphoma

V(D)J recombination generates a hugely diverse repertoire of antigen receptors through the random joining of antigen receptor gene segments. Recombination is catalysed by the RAG proteins which bind to and cleave recombination signal sequences (RSSs) that flank each coding segment. Since this reaction involves the introduction of double-strand breaks in the genome, it poses a major risk to genome stability. Indeed, 35-40% of lymphoid cancers have chromosome translocations that bear the hallmarks of mistakes in the production of antigen receptor genes. Recently, V(D)J recombination was proposed to trigger genome instability by a new mechanism called reintegration, where the by-product of recombination, the excised signal circle (ESC), is reinserted back into the genome at an RSS. I examined the cleavage step of reintegration and found that an ESC stimulates cleavage of an RSS, but not vice versa. For reintegration to occur, simultaneous cleavage at an RSS and ESC is required, so this finding suggests that reintegration may not occur as frequently as others have predicted. Instead, the ESCs could trigger double-strand breaks at RSSs and cryptic RSSs throughout the genome, whilst remaining intact after each cleavage event, in a proposed new mechanism called “cut-and-run”. I then investigated the molecular basis of asymmetric cleavage, and found that it is most likely caused by RAG proteins binding to both sides of an ESC simultaneously and obscuring the cleavage site. Using transfected extrachromosomal substrates, I found that RSS-ESC cleavage is also asymmetric in vivo. Finally, analysis of an endogenous ESC revealed that it is largely intact in a pro-B cell line, consistent with the cut-and-run hypothesis. Therefore, cut-and-run could be a potent source of genomic instability in developing lymphocytes that creates broken ends for chromosome translocations.