Synergistic requirement of orphan nonamer-like elements and DNA bending enhanced by HMGB1 for RAG-mediated nicking at cryptic 12-RSS but not authentic 12-RSS

Abstract

V(D)J recombination is initiated by the specific binding of the recombination activating gene (RAG) complex to the heptamer and nonamer elements within recombination signal sequence (RSS). The break points associated with some chromosomal translocations contain cryptic RSSs, and mistargeting of RAG proteins to these less conserved elements could contribute to an aberrant V(D)J recombination. Recently, we found RAG-dependent recombination in the hotspots of TEL-AML1 t(12;21)(p13;q22) chromosomal translocation by an extrachromosomal recombination assay. Here, we describe using in vitro cleavage assays that RAG proteins directly bind to and introduce nicks into TEL and AML1 translocation regions, which contain several heptamer-like sequences. The cryptic nicking site within the TEL fragment was cleaved by RAG proteins essentially depending on a 12-RSS framework, and the nicking activity was enhanced synergistically by both HMGB1 and orphan nonamer-like (NL) sequences, which do not possess counterpart heptamers. In addition, we found that DNA bending stimulated by HMGB1 is indispensable for the HMGB1- and orphan NL element-dependent enhancement of RAG-mediated nicking at the cryptic 12-RSS. Collectively, we would propose the mechanism of HMGB1-dependent enhancement of RAG-mediated nicking at a cryptic RSS through enhanced DNA bending.