HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

We show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn2+, but not Mg2+. Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg2+ is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg2+ can be substantially rescued when Mg2+ is replaced with Mn2+.The conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg2+ in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS.Antigen receptor genes are assembled from component variable (V), diversity (D), and joining (J) gene segments through a site-specific DNA rearrangement process called V(D)J recombination (for reviews, see [1,2]). This process is initiated by two lymphoid cell-specific proteins, called RAG1 and RAG2 (recombination activating genes-1 and -2), which collaborate to bring two gene segments into close proximity by establishing protein-DNA contacts with a conserved recombination signal sequence (RSS) that flanks each gene segment. The RAG proteins subsequently catalyze the formation of a DNA double-strand break at the junction between the coding segment and the RSS through a two-step nick-hairpin mechanism. Each RSS contains a highly conserved heptamer and nonamer element separated by relatively nonconserved spacer DNA that is typically either 12 or 23 bp in length (12-RSS or 2