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Abstract
BackgroundV(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.ResultsHere we identify a novel DNA breakpoint site in the plasmid V(D)J recombination substrate pGG49 (bps6197) that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3') but contains a nonamer-like element (5'-ACATTAACC-3') 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity.ConclusionWe have identified a novel DNA sequence, called bps6197, that supports standard V(D)J-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires the presence of HMGB1/2, is stimulated by synapsis with a 12-RSS partner, and is directed in part by an inverted repeat sequence adjacent to the DNA cleavage site. These results have important implications for understanding how the RAG proteins can introduce a DNA double-strand break at DNA sequences that do not contain an obvious heptamer-like motif.
Highlights
V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the Recombination Activating Gene-1 (RAG1) and Recombination Activating Gene-2 (RAG2) proteins via a nick-hairpin mechanism
Identification of a novel RAG-mediated breakpoint sequence, bps6197, in pGG49 that lacks an obvious heptamer In a previous study, Raghavan and Lieber characterized the V(D)J recombination potential of several putative cRSSs identified from lymphoid malignancies, including Ttg-1 and Hox11 [4]
Consistent with previous results, we find that signal end breaks (SEBs) are readily detected in plasmid substrates containing a consensus 23-RSS when incubated with wild-type but not catalytically inactive RAG proteins (WT and D600A cMR1/cMR2, respectively) in the presence of High Mobility Group Box 1 (HMGB1), but the abundance of these breaks diminishes when the 23-RSS is replaced by the Ttg-1 sequence, and is reduced even further upon replacement with the Hox11 sequence (Fig. 1B)
Summary
V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element. To be considered a plausible cRSS, it is generally thought that the putative cRSS must minimally contain at least the first three residues of the consensus heptamer (CAC) [9] due to the high degree of sequence conservation of these residues among bona fide RSSs [10] and based on functional studies of mutant RSS substrates which demonstrate that mutation of any of these residues essentially abolishes RAG-mediated cleavage [11,12,13] and V(D)J recombination [10]. Whether cellular factors can help stabilize alternative DNA conformations in otherwise complementary DNA which can be targeted for RAG-mediated cleavage remains unclear
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