HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex
© Zhang and Swanson; licensee BioMed Central Ltd. 2009
Received: 21 November 2008
Accepted: 24 March 2009
Published: 24 March 2009
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. 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.
Here 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.
We 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.
Antigen receptor genes are assembled by a process known as V(D)J recombination from arrays of variable (V), diversity (D), and joining (J) gene segments that are flanked on one (V, J) or both (D) sides by a conserved recombination signal sequence (RSS) (for reviews, see [1–3]. The RSS contains a conserved heptamer element (consensus: 5'-CACAGTG-3') separated by either 12 or 23 base pairs (bps) from a conserved nonamer element (consensus: 5'-ACAAAAACC-3'). The RSS functions as the binding site for the RAG1 and RAG2 proteins, which together initiate V(D)J recombination by cleaving the RSS at the 5'-end of the heptamer through a two-step nick-hairpin mechanism. Normally, recombination only occurs between two gene segments that differ in the spacer length of the flanking RSS (one 12-RSS and one 23-RSS), a restriction termed the 12/23 rule. However, studies of recurrent breakpoint sequences identified in some types of lymphoid malignancies suggest that the RAG proteins may occasionally mediate illegitimate recombination by introducing a site-specific DNA break at a sequences resembling an RSS (a "cryptic" RSS or cRSS) [4–6], or catalyzing structure-specific cleavage at sites prone to adopting non-B form DNA conformations [7, 8].
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)  due to the high degree of sequence conservation of these residues among bona fide RSSs  and based on functional studies of mutant RSS substrates which demonstrate that mutation of any of these residues essentially abolishes RAG-mediated cleavage [11–13] and V(D)J recombination . The RAG proteins also exhibit structure-specific nicking of DNA, preferentially targeting transitions from single- to double-stranded DNA [7, 11, 12, 14, 15]. Nicks can lead to DNA double strand breaks if they are introduced on both DNA strands in close proximity. To study structure-specific nicking by the RAG proteins, most studies have artificially introduced transitions from single- to double-stranded DNA into DNA substrates by incorporating bp mismatches, bulges, flaps, or gaps. Only one example has been reported of an otherwise fully complementary double-stranded DNA adopting a non-B form DNA conformation that is targeted by the RAG complex for nicking . Whether cellular factors can help stabilize alternative DNA conformations in otherwise complementary DNA which can then be targeted for RAG-mediated cleavage remains unclear.
In principle, h igh m obility g roup proteins that belong to the HMG-b ox family of architectural DNA binding and bending factors (e.g. HMGB1 or HMGB2) are plausible candidates for promoting mistargeted RAG activity because they are capable of binding structurally distorted DNA , such as four-way junctions  and damaged or modified DNA [18–20], and also can interact directly with the RAG proteins  and stimulate RAG-mediated RSS binding and cleavage activity in vitro [22, 23] and V(D)J recombination in cell culture assays . In support of this possibility, we have identified 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 in the presence of HMGB1/2. We find that RAG-mediated bps6197 cleavage yields a blunt end with the sequence 5'-CCTGACG-3' that is separated by 23-bps from a nonamer-like element (5'-ACATTAACC-3'). RAG cleavage activity at bps6197 is stimulated by synapsis with a 12-RSS, and requires both HMG-box domains of HMGB1 to be intact. Evidence is presented that HMGB1/2 targets a dyad-symmetric inverted repeat sequence lying 5' of the cleavage site. The implications of these findings are discussed.
Identification of a novel RAG-mediated breakpoint sequence, bps6197, in pGG49 that lacks an obvious heptamer
RAG-mediated cleavage of bps6197 occurs through a nick-hairpin mechanism and is stimulated by synapsis with a 12-RSS
To follow up this experiment, we incubated the substrates with WT or D600A cMR1/cMR2 in the absence or presence of HMGB1 and analyzed the reaction products on a denaturing polyacrylamide gel in parallel with native gel-isolated cleavage products A and B as well as sizing markers derived from restriction endonuclease cleavage of the intact bps6197/12/23 substrate (see Fig. 2A). We find that cleavage product B migrates more slowly than a 236 nt fragment generated by Sal I digestion of the intact bps6197/12/23 substrate, and comigrates with the major reaction product generated by RAG-mediated cleavage (Fig. 2C, compare lane 4 to lanes 7 and 8). In contrast, cleavage product A migrates just moderately faster than the Sal I cleavage product, but much more slowly than a 95 nt product generated by Aat II digestion (not shown, but see Fig. 4 for example), and is only observed in the in vitro cleavage reactions containing WT cMR1/cMR2 and HMGB1 (Fig 2C, compare lane 3 to lanes 7 and 8). Moreover, the abundance of this product increases when the 23-RSS is replaced by Ttg-1 or Hox11 sequences, and this increase is correlated with reduced nicking at these cRSSs (Fig. 2C, compare lane 8 to lanes 11 and 14). Faster migrating reaction products are also detected that correspond to nicks introduced at positions 6195 and 6197 (see Additional File 1: Verification of nicking sites in PCR-generated substrates containing bps6197), with the latter product being more abundant than the former. The apparent doubling in the size of product A between native and denaturing gels and the detection of nicks at bps6197 provide compelling evidence that bps6197 supports RAG-mediated cleavage by a nick-hairpin mechanism.
The relationship between the level of nicking observed at the 23-RSS/cRSS and bps6197 cleavage activity suggested that the removing the 23-RSS itself may be sufficient to promote bps6197 cleavage. To test this possibility, and the role of the 12-RSS in supporting bps6197 cleavage activity, mutant substrates were prepared that lacked one or both RSSs (Fig. 2A) and then these substrates were subjected to in vitro cleavage by WT or D600A cMR1/cMR2 in the absence or presence of HMGB1 (Fig. 2C). We find that substrates lacking the 12-RSS support lower levels of bps6197 hairpin formation than their counterparts containing a 12-RSS (Fig. 2C, compare lanes 8, 11, and 14 to lanes 17, 20, and 23), but removal of the 23-RSS enhanced bps6197 cleavage relative to the 6197/12/23 substrate, with substrate cleavage levels similar to that observed with the 6197/12/Hox11 substrate (Fig 2C, compare lane 26 to lanes 8 and 14). In contrast, when both the 12- and 23-RSS were absent, bps6197 was cleaved poorly (Fig. 2C, lane 29). Taken together, these data strongly suggest that bps6197 cleavage is stimulated by synapsis with a 12-RSS partner. To determine whether the distance or orientation of the 12-RSS partner positioned in cis influences the efficiency of RAG-mediated bps6197 cleavage, we removed the proximal 12-RSS and replaced the distal 23-RSS with a 12-RSS in either orientation. We find that the level of RAG-mediated bps6197 cleavage of these substrates in vitro is quite similar to that observed with the 6197/12 substrate (see Additional File 2: RAG-mediated bps6197 cleavage is not affected by the distance or orientation of the 12-RSS partner), suggesting that RAG-mediated bps6197 cleavage is not affected by the distance or orientation of the 12-RSS partner.
Analysis of RAG-mediated cleavage and binding of bps6197 using oligonucleotide substrates
In principle, the lower cleavage activity observed with bps6197 relative to a consensus 23-RSS could reflect poorer RAG binding to this substrate. To test this possibility, we compared the RAG binding activity between the consensus 23-RSS and bps6197 substrates using an electrophoretic mobility shift assay (EMSA). In the first experiment, radiolabeled substrates were incubated with WT cMR1/cMR2 alone or with HMGB1 in the absence or presence of increasing amounts of cold 23-RSS as a specific competitor (Fig. 3B, left panel). In the absence of HMGB1, WT cMR1/cMR2 assembles two discrete protein-DNA complexes with a consensus 23-RSS. We have previously shown that these RAG-RSS complexes, called SC1 and SC2, both contain a RAG1 dimer and either one (SC1) or two (SC2) molecules of RAG2 . Both complexes are supershifted by the addition of HMGB1, and the abundance of these complexes is diminished by further addition of increasing amounts of cold 23-RSS competitor. We find that the RAG proteins assemble the various protein-DNA complexes on the bps6197 substrate similarly to a consensus 23-RSS, with protein-DNA complex formation only slightly more susceptible to competition by a cold 23-RSS. To confirm these results, we assembled RAG complexes on a radiolabeled 23-RSS substrate in the presence of increasing concentrations of cold 23-RSS, bps6197, Hox11 or non-specific competitor DNA (Fig. 3B, right panel). We find that, as competitors, the substrates can be ordered from strongest to weakest as follows: 23-RSS>bps6197>Hox11>non-specific DNA, with differences between each being approximately 2–3 fold. Together, these data suggest that bps6197 is competent to support stable formation of RAG-DNA complexes in vitro to levels that are close (within ~2-fold) to a consensus 23-RSS.
Site-specific bps6197 cleavage is directed by inverted repeat and nonamer-like sequences
Determinants of HMGB1 required to promote RAG-mediated bps6197 cleavage
The dependence of RAG-mediated bps6197 cleavage in long DNA fragments on HMGB1 led us to question what determinants of HMGB1 were required to support this activity. HMGB1 and its vertebrate homologue HMGB2, contain tandem HMGB-box domains (called A and B) and an acidic C-terminal tail separated from the HMG-boxes by a linker sequence rich in basic residues . Both HMG-box domains share a globally similar architecture comprised of three alpha helices that adopt an L-shaped structure. Both domains possess DNA binding activity, primarily interacting with the minor groove and mediating DNA bending in part by intercalating hydrophobic residues between DNA bps. Despite this similarity, the two domains exhibit distinct DNA binding preferences: box A selectively binds distorted DNA structures, whereas box B shows less preference for such structures but, unlike box A, can itself induce a severe bend in linear DNA. The functional activity of the HMG-box domains is further modulated in various ways by the C-terminal basic and acidic residues.
Bps6197 supports enhanced binding by HMGB1 compared to a 23-RSS
Cleavage and recombination activity of bps6197 in cells
Having established that the RAG proteins can cleave bps6197 when embedded in the pJH299 backbone, we next cotransfected these substrates with WT or D600A cMR1 and WT cMR2 expression constructs in 293 cells and analyzed plasmid DNA recovered 72 h after transfection for bps6197 and/or RSS SEBs. We find that SEBs at both RSSs are readily detected in the recovered 12/23 substrate cotransfected with WT cMR1/cMR2, but not D600A cMR1/cMR2 (Fig. 8B, bottom panel). However, in contrast to in vitro cleavage assays, bsp6197 SEBs are not observed in the 12/6197SO plasmid DNA recovered from cell culture, despite detecting cleavage at the 12-RSS positioned in cis (which occurs at lower levels than in the 12/23 substrate). We considered the possibility that HMGB1 expression may be insufficient in 293 cells to promote RAG-mediated bps6197 cleavage. However, HMGB1 is readily detected in 293 cell lysates and repeating these assays in 293 cell lines overexpressing HMGB1 did not change the outcome of the experiment (data not shown). Therefore, we conclude that bps6197 is not cleaved by the RAG proteins in cells to levels that are within the detection limit of our LM-PCR assay. We consider possible explanations for this result below.
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. 1A). Close inspection of Figure 1B 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. 2C), 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 . 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  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 . 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 , as HMGB1 alone, despite displaying some sequence preference for bps6197 over the 23-RSS, nevertheless binds this site relatively weakly (Fig. 7). 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, 29].
We have uncovered an example of illegitimate DNA cleavage by the RAG proteins at a DNA sequence called bps6197 that lacks an apparent heptamer motif. We demonstrate that bps6197 functionally resembles a 23-RSS and that RAG-mediated cleavage at this site: (i) occurs through a nick-hairpin mechanism; (ii) depends on the presence of HMGB1/2; (iii) is stimulated by synapsis with a 12-RSS; and (iv) is partly directed by an inverted repeat sequence 5' of the breakpoint. We provide evidence that HMGB1 alone binds bps6197 better than a 23-RSS, and that efficient RAG-mediated cleavage of this site requires both HMG-box domains to be intact. Taken together, these data suggest that HMGB1/2 can target illegitimate RAG-mediated cleavage at sequences lacking an evident heptamer. These results raise the possibility that breakpoint sequences identified in lymphoid malignancies that were previously thought unlikely to undergo RAG-mediated cleavage due to the absence of a cRSS with a heptamer-like sequence may nevertheless support cleavage by the RAG proteins in the presence of cofactors like HMGB1/2.
RAG and HMGB1 protein purification
Truncated and full-length forms of RAG1 (residues 384–1040 and 1–1040, respectively) and RAG2 (residues 1–387 and 1–517, respectively) containing an amino-terminal maltose binding protein fusion partner (cMR1, FLMR1, cMR2, and FLMR2, respectively) were coexpressed in 293 cells and purified by amylose affinity chromatography according to published procedures . Full-length, truncated, or mutant forms of amino-terminal polyhistidine-tagged HMGB1 were expressed in E. coli and purified as described elsewhere .
Oligonucleotide substrates containing a 12-RSS or 23-RSS cRSS labelled at the 5' end of the top strand were prepared as described elsewhere [24, 30]. A substrate containing the bps6197 sequence was similarly prepared from the oligonucleotide 5'-TCCCCGAAAAGTGCCACCTGACG TCTAAGAAACC ATTATTATCATGACATTAACC TATAAA-3' and its complement (positions equivalent to the 23-RSS heptamer and nonamer motifs are underlined). Derivatives of this substrate containing mutations at positions indicated in the text were also prepared. Unlabeled DNA substrates containing consensus 12-RSS, 23-RSS, Hox11 or non-specific DNA sequences (DAR81/82) were prepared from oligonucleotides described previously [11, 24].
The plasmid V(D)J recombination substrates pGG49, its derivatives containing Ttg-1 or Hox11 cRSS sequences, and pJH299 have been described elsewhere [4, 10]. Derivatives of pGG49 lacking the 12-RSS or 23-RSS were generated by Sal I or BamH I digestion, respectively. Variants of pGG49 and pJH299 containing the wild-type or mutant bps6197 sequences discussed in the text were generated according to procedures described in Additional File 3: Supplementary Materials and Methods. Long DNA fragments (~650 bp) radiolabeled at the 5' end of the top strand containing the RSS, cRSS, and/or wild-type or mutant bps6197 sequences present in pGG49 and its derivatives were generated by PCR using radiolabeled primer 6000F (5'-TATTGTCCTCATGAGCGGATAC-3') and unlabeled primer 6624R (5'-GAACGGTGGTATATCCAGTG-3'). PCR samples (100 μl final volume) containing plasmid template DNA (70 ng) and primers (20 pmol each) were subjected to initial denaturation at 95°C for 4 min, followed by 30 cycles of amplification (95°C for 45 sec, 56°C for 30 sec, and 72°C for 60 sec) and a final extension (72°C for 4 min). PCR products were isolated using a QIAquick PCR purification kit (Qiagen), eluted in 50 μl buffer EB (10 mM Tris, pH 8.0), fractionated on a vertical agarose gel (1.4%) at 150V for 1.5 h, and gel-isolated DNA purified using a QIAquick Gel Extraction Kit (Qiagen).
In vitro RAG cleavage and binding assays
RAG-mediated cleavage of oligonucleotide substrates, PCR-generated long DNA fragments, or plasmid DNA was analyzed using an in vitro cleavage assay described previously . Unless otherwise noted, basic in vitro cleavage reactions (10 μL) contained cMR1/cMR2 (~100 ng) and substrate DNA (~0.02 pmol radiolabeled oligonucleotide, ~250 ng long DNA substrate or100 ng unlabeled Bgl II-digested plasmid DNA) in reaction buffer containing Mg2+. Reactions were further supplemented with various forms of HMGB1 without or with additional unlabeled 12-RSS or 23-RSS partner (1 pmol) as indicated. Cleavage reactions were incubated at 37°C for 1 h. For some reactions containing long radiolabeled DNA fragments (e.g. Fig. 2B), DNA was purified using a QIAquick PCR purification kit, and reaction products separated under native conditions on a vertical 1% agarose gel. For other samples containing radiolabeled DNA, reactions were terminated by adding 2 volumes of sample loading solution (95% formamide, 10 mM EDTA) and reaction products fractionated under denaturing conditions on a 15% polyacrylamide sequencing gel lacking formamide (oligonucleotides) or an 8% polyacrylamide gel containing 40% formamide (long DNA fragments). Reaction products were analyzed using a Storm 860 phosphorimager running the ImageQuaNT software. For reactions containing unlabeled plasmid DNA (e.g. Fig. 1B), samples were diluted by adding 20 μL TE (10 mM Tris [pH 8.0], 1 mM EDTA], and heat inactivated for 10 min at 65°C. From 4 μL of this sample, SEBs generated at the 23-RSS or its replacement were detected using LM-PCR as previously described [24, 31]. The formation of RAG and HMGB1 protein-DNA complexes on oligonucleotide substrates was analyzed using an EMSA as previously described .
Recombination Activating Gene-1
Recombination Activating Gene-2
High Mobility Group Box 1
High Mobility Group Box 2
breakpoint sequence 6197
electrophoretic mobility shift assay
This research was supported by grants to PCS from the Nebraska LB692 Tobacco Settlement Biomedical Research Program and from the NIH (R56AI55599-A1), and was conducted in laboratories renovated with support from the Research Facilities Improvement Program of the NIH National Center for Research Resources (C06 RR17417-01). The authors wish to thank Megan Lewandowski for technical assistance.
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