Abstract
BackgroundRAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction.ResultsWe 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+.ConclusionThe 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.
Highlights
Recombination Activating Gene-1 (RAG1) and Recombination Activating Gene-2 (RAG2) initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment
We find that individual HMG-box domains stimulate RAG-mediated cleavage of a single RSS in Mn2+, but not Mg2+; this observation is consistent with and largely reconciles the conflicting data published by Bergeron et al and Dai et al Interestingly, when these assays are repeated under conditions favouring synapsis, we find that individual HMG-box domains gain the ability to stimulate RAG-mediated RSS cleavage
No decrease in aberrant 23-RSS nicking by the RAG complex is observed. When this experiment was repeated using buffer containing Mn2+, we find that, in contrast to results obtained in Mg2+, the RAG proteins support modest 23-RSS substrate cleavage in the absence of High Mobility Group Box 1 (HMGB1) (Fig. 2B, lane 2), but both full-length and individual HMG-box domains exhibit a concentration-dependent stimulation of RAGmediated 23-RSS cleavage (Fig. 2B, lanes 3–6, 11–14, and 19–22)
Summary
RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. High mobility group proteins that belong to the HMG-box family of architectural DNA binding and bending factors (e.g. HMGB1 or HMGB2) were later found to stimulate RAG binding and cleavage of isolated RSSs ( the 23-RSS), and facilitate synapsis and coupled cleavage of RSS pairs according to the 12/23 rule of V(D)J recombination [4,5]. A short basic linker connects box B to a C-terminal acidic tail containing about 30 contiguous aspartate and glutamate residues While both HMG-box domains interact with DNA, they exhibit distinct DNA binding properties: whereas box A prefers to bind structurally distorted DNA, box B lacks this selectively, but can itself induce severe bends into linear DNA, which is a property box A lacks [8,9,10,11]. The DNA binding activity and functional properties of the HMG-box domains are strongly influenced by flanking basic and acidic regions of these proteins [11,12,13,14,15]
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