Here we describe the identification of a novel breakpoint sequence in pGG49 called bsp6197 that is targeted for RAG-mediated cleavage in vitro in the presence of HMGB1. Because the sequence lacks an obvious heptamer motif and possesses an inverted repeat in the sequence flanking the breakpoint, we were curious about the mechanism underlying the cleavage reaction and considered the possibility that RAG-mediated cleavage is directed by the inverted repeat. We show here that bps6197 supports RAG-mediated cleavage via a nick-hairpin mechanism. Efficient RAG-mediated bps6197 cleavage depends on the presence of HMGB1 and synapsis with a partner 12-RSS, and is guided in part by the inverted repeat sequence. To our knowledge, this is the first example of a flanking inverted repeat sequence influencing where the RAG proteins initiate DNA cleavage.
Given the dependence of RAG-mediated bps6197 cleavage on synapsis with a 12-RSS, we might have expected to detect LM-PCR products resulting from linker ligation to SEBs at both bps6197 and the 12-RSS, using the linker primer as both a forward and reverse primer. The predicted size of this LM-PCR product is 236 bp, which is 24 bp longer than the LM-PCR product that identifies the 23-RSS SEB using the linker primer and the primer downstream of the 23-RSS (23P, see Fig. ). Close inspection of Figure does show the presence of low levels of LM-PCR products running slightly larger than those detecting the 23RSS SEB that are consistent with the introduction of SEBs at both the 12-RSS and bps6197. Their abundance may be less than one might expect for several possible reasons, including a lower probability of linker ligation at both SEBs, amplification from substrates containing bps6197 SEBs associated with a nicked 12-RSS (which would not be detected on the denaturing gels in Fig. ), and/or competition with PCR products amplified using the linker primer and 23P primer.
In our previous study, we found that RAG-mediated nicking and cleavage of oligonucleotide substrates containing cRSSs identified from lymphoid malignancies were either unchanged or slightly reduced in the presence of HMGB1 for most cRSSs tested (including LMO2, TAL1, Hox11, SIL, SCL), except Ttg-1 [24
]. In the latter case, nicks at the predicted heptamer (which contains a fully consensus sequence) was not enhanced by addition of HMGB1, but nicks at other locations in the Ttg-1 sequence increased when HMGB1 was added to the cleavage reaction. In contrast, RAG-mediated nicking of a bps6197 oligonucleotide substrate increases with addition of HMGB1, and efficient nicking and hairpin formation at this site in long DNA is HMGB1-dependent.
One possible reason why RAG-mediated bps6197 cleavage shows a more stringent requirement for HMGB1 than other cRSSs examined previously is that HMGB1 may target and/or stabilize a structural distortion in the flanking inverted repeat sequence. This possibility is made plausible by previous studies showing that HMGB1 binds four-way junctions [17
] and is further supported by evidence presented here that HMGB1 binds bps6197 better than a consensus 23-RSS. However, attempts to detect a stable pre-existing or protein-induced four-way junction in the inverted repeat of bps6197 using DNA footprinting experiments failed to yield compelling evidence for the existence of such a structure (data not shown), but we point out that this may be difficult to detect due to its small size, or if the structure is transient, limited in abundance, or masked in the bound complex. Similarly, although the lack of detectable bps6197 cleavage in cell culture experiments could be interpreted to mean that the RAG proteins fail to recognize this sequence in vivo
, it is possible that factors bound to the plasmid substrate in cells render the site inaccessible to the RAG and/or HMGB1/2 proteins or unable to adopt a conformation that is targeted by HMGB1/2.
Although authentic RSSs in antigen receptor loci are the normal targets of the RAG proteins during V(D)J recombination, illegitimate RAG activity has been implicated in the etiology of chromosomal abnormalities recurrent in certain forms of leukemia and lymphoma [9
]. A subset of these events have been attributed to the RAG proteins mistargeting a sequence resembling an RSS, and mediating a standard V(D)J-type rearrangement between an authentic RSS and a cRSS. In cases where there is clear evidence for this type of rearrangement, the cRSS contains at least the first three residues of the consensus heptamer (5'-CAC). A second subset of events has been suggested to occur through the illegitimate repair of a mechanistically undefined DNA double strand break with DNA ends produced by RAG-mediated cleavage at a pair of authentic RSSs. The source of the undefined DNA break in the second type of recombination event is unclear as these sites generally do not have a recognizable cRSS with a plausible heptamer motif. The data presented here raise the possibility that such sites, despite lacking an obvious heptamer, could nevertheless be subjected to illegitimate RAG-mediated cleavage if they contain a nonamer-like sequence that could anchor the RAG complex in proximity to the breakpoint. By recognizing or stabilizing a structural distortion at the breakpoint, HMGB1/2 may then promote illegitimate DNA cleavage by targeting the anchored RAG complex to the breakpoint in lieu of a consensus heptamer. It is likely that stable HMGB1/2 association with the breakpoint depends on concomitant interactions with the RAG proteins themselves, most likely RAG1 [21
], as HMGB1 alone, despite displaying some sequence preference for bps6197 over the 23-RSS, nevertheless binds this site relatively weakly (Fig. ). This targeting mechanism need not be unique to HMGB1/2, as it could also be facilitated directly or indirectly by transcription factors that bind in or near the breakpoint sequence and interact with the RAG proteins as an illegitimate form of an otherwise normal process [28