Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA

Abstract

RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs. RAG nucleases play a critical role in the generation of T- and B-cell receptor diversity through splicing different sub-exons of these receptors together. Although RAG-mediated DNA recombination is guided by specific recognition sequences, this complex can also act as a structure-specific nuclease to promote genomic instability, potentially leading to the development of lymphoma and leukemia. Here, Kumari and Raghavan explore DNA structural and sequence factors that influence this potentially pathological activity of RAGs.