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Previous Article | Next Article 
Molecular and Cellular Biology, June 2005, p. 4413-4425, Vol. 25, No. 11
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.11.4413-4425.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Ben Wong,2,
Yi-Meng Yen,2
Marjorie A. Oettinger,1*
Jongbum Kwon,1,
and
Reid C. Johnson2*
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114,1 Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095-17372
Received 16 January 2005/ Returned for modification 15 February 2005/ Accepted 1 March 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both the binding of RAG1/2 to a single RSS and formation of the 12/23 RSS synaptic complex have been shown to be strongly enhanced by HMGB1/2 proteins (see references 13 and 15 and references therein). Formation of the RAG1/2-HMGB-RSS complex is followed by the hydrolysis of one strand of DNA, leaving a 3' hydroxyl group at the border of the coding sequence (Fig. 1A). The free hydroxyl of this nicked DNA then attacks the 5' phosphate of its base-paired partner on the antiparallel strand, creating a hairpin coding end and a blunt, 5'-phosphorylated signal end (30). These chemical steps are believed to require changes in the structure of DNA within the RSS, particularly over the heptamer region and flanking coding DNA (14). HMGB proteins are ubiquitously present in all eukaryotic organisms and possess one or more structurally conserved HMGB DNA binding domains (for reviews see references 3, 9, 16, 53, and 54). Each domain has 75 to 80 amino acids and consists of an extended strand plus three 
-helices that fold into an L-shaped structure (Fig. 1B to D). Members of the sequence-specific class of HMGB proteins, such as LEF1 and SRY, are usually transcription factors whose expression is cell type specific. The nonsequence-specific HMGB proteins, such as HMGB1/2 in mammals and Nhp6A in Saccharomyces cerevisiae, are among the most abundant nonhistone chromatin-associated proteins. Mammalian HMGB1 is found throughout most adult mouse tissues except for regions within the brain (9, 18), whereas HMGB2 is restricted to lymphoid tissues and the testis (44).
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Mammalian HMGB1 efficiently stimulates cleavage by the RAG1/2 proteins, while the yeast HMGB protein Nhp6A exhibits poor but detectable activity, and an unrelated prokaryotic DNA bending protein HU is inactive (57). The poor activity of Nhp6A could be due to (i) the presence of only a single HMGB domain, (ii) an intrinsic difference in the DNA binding properties of Nhp6A and HMGB1, or (iii) the absence of a direct protein-protein interaction between the yeast protein and RAG1/2 that is important for HMGB1 activity in the V(D)J reaction. In support of the last possibility, Aidinis et al. presented evidence that HMGB1 and RAG1 interact in solution and that this interaction requires both HMGB domains (4).
Here we show that individual domains of HMGB1 efficiently assemble cleavage-competent RAG1/2-RSS complexes on naked DNA but that both domains are required for maximal activity when the RSS is associated with a nucleosome. The surface-exposed phenylalanine at the DNA exit wedge of HMGB proteins, a residue critical for recognition of a preexisting DNA kink (19, 61), is essential for stimulating DNA binding and cleavage by RAG1/2. Surprisingly, we find that although Nhp6A efficiently stimulates the binding of RAG1/2 to the RSS, these RAG1/2-RSS complexes do not support DNA cleavage. Thus, the requirements for stimulating binding can be separated from those needed for promoting catalysis. Additional analysis of Nhp6A mutants revealed that those derivatives which bind prebent DNA less stably than the wild type are able to stimulate both RAG1/2-RSS binding and V(D)J cleavage. Taken together, these results identify the functionally important determinants of HMGB proteins for the assembly of catalytically active RAG1/2-RSS complexes and indicate that a dynamic protein-DNA complex is required for RAG-mediated DNA cleavage.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dATP as described previously (40, 63). Substrates for microcircles were internally labeled PCR products that were purified using QIAGEN PCR purification columns, digested with 100 units of EcoRI (New England Biolabs) for 3 h at 37°C, extracted with phenol-chloroform, and ethanol precipitated. DNA microcircle ligation assays were performed as described previously (40, 63). Remaining linear DNA substrates were removed by digestion with exonuclease III (New England Biolabs), and the samples were then incubated with proteinase K in the presence of 0.5% sodium dodecyl sulfate followed by extraction with phenol-chloroform. The amount of monomeric microcircles formed was quantitated by phosphorimaging (ImageQuant; Amersham-Pharmacia) after electrophoresis in 7% acrylamide:bisacrylamide (59:1) gels containing 10% glycerol and Tris-borate-EDTA buffer. For microcircle binding assays, preformed 98-bp 32P-labeled microcircles were prepared using HMGB1 and T4 DNA ligase. Binding reactions were performed under the same conditions as for the linear substrates. For competition assays, sufficient HMGB proteins were incubated with 98-bp microcircles in a 20-µl reaction volume containing 20 mM HEPES (pH 7.5), 40 mM NaCl, 1 mM EDTA, 0.1 mg/ml acetylated bovine serum albumin (BSA) (Roche), and 5% glycerol for 30 min at 30°C to generate at least 50% complexes. From 0 to 3.3 µg sonicated salmon sperm DNA (Sigma) was then added, and the reaction mixtures were incubated an additional 30 min at 30°C and applied to a polyacrylamide gel. For dissociation rate experiments, the complexes were challenged with 55 µg/ml sonicated salmon sperm DNA (Pharmacia), which corresponds to approximately a 1,500-fold mass excess of competitor DNA, and aliquots were loaded onto electrophoresing gels at increasing times. Gels were dried and subjected to phosphorimaging. Dissociation rates reflect the times required for decay of 50% of the starting complex, as measured from the times at which the samples were applied to the gel, and were extrapolated from graphs like those in Fig. 6B.
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(ii) Single-site cleavage. Plasmids12RSS/TP3 and 23RSS/TP2 (25) served as the sources for the DNA substrates for single-site cleavage. 32P-labeled substrate DNA was prepared as described previously (25). The single-site cleavage reactions were performed in a 20-µl final volume containing 1.5 ng labeled substrate DNA, 20 ng His-tagged core RAG1, 20 ng core RAG2, 1% BSA, 21 mM HEPES (pH 7.5), 1.7 mM DTT, 62.5 mM K-glutamate, 2 mM MnCl2, and 20 ng fragmented and boiled herring sperm DNA. The use of Mn2+ as the divalent metal ion permits cleavage in the presence of only a single RSS. Reaction mixtures were incubated for 2 h at 30°C. Reaction products were separated on a 6% denaturing acrylamide:bisacrylamide (19:1) gel, visualized by autoradiography, and quantified with a phosphorimager using ImageQuant software.
(iii) Nucleosome cleavage assays. Nucleosome cleavage assays were carried out essentially as described previously (25, 26). Nucleosomes were assembled by the salt dilution method using bulk acetylated histones and a 152-bp DNA template (12/TP3 or 23/TP2) and then purified through a 5 to 30% glycerol gradient. Equal amounts of free DNA and nucleosomes (using equal 32P activities equivalent to 1 ng) were used in 20-µl cleavage reaction mixtures as described previously (25, 26).
(iv) Gel shift assay. Electrophoretic mobility shift assay reactions were carried out as previously described (34) using Ca2+ as the divalent metal ion to allow the RAG proteins to bind to but not cleave their substrate DNA. In brief, binding reaction mixtures in a final volume of 10 µl contained 0.75 ng labeled substrate DNA, 10 ng maltose-binding protein-core RAG1, 10 ng core RAG2, 20 mM HEPES (pH 7.5), 55 mM K-glutamate, 2 mM CaCl2, 1.6 mM DTT, 1% glycerol, 1% BSA, and 0.3 ng herring sperm DNA. The amount of HMGB protein present in each reaction mixture is indicated in the figure legends. Binding was allowed to proceed at 30°C for 30 min, and the products separated on a 5% polyacrylamide gel (29:1) (0.5x Tris-borate-EDTA and 5% glycerol) at 250 V for 3 h.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of RAG1/2-RSS binding and DNA cleavage by truncated HMGB1 derivatives. While RAG1/2 alone can bind to an individual RSS, HMGB1 enhances this binding, promoting efficient formation of a complex that migrates more slowly than the RAG1/2-RSS complex formed in the absence of HMGB1 (for examples, see references 45 and 57 and Fig. 3A). We found that the individual HMGB domains are sufficient to stimulate formation of high levels of RAG1/2 complexes on either the 12 or 23 RSS (Fig. 3A), although more domain B' than domain A' protein is required to generate an equivalent amount of the slower-migrating RAG1/2-HMGB-RSS complex (Fig. 3A). HMGB1 does not require the presence of the C-terminal charged extensions to form the RAG1/2-HMGB-RSS complex. Derivatives AB', AB, A, and B all are capable of forming high levels of slower-migrating complexes (Fig. 3A and data not shown). The domain B" derivative was ineffective, consistent with its very poor DNA binding and microcircle formation activities (not shown).
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Stimulation of RAG1/2 activity on nucleosomal substrates by HMGB1 derivatives. Previous studies have shown that nucleosomal substrates are refractory to V(D)J cleavage. This inhibition can be relieved in part through acetylation of the nucleosomes and the addition of HMGB1, particularly if the RSS is positioned near the entry/exit points of the nucleosome (25, 26). The ability of different HMGB1 derivatives to support cleavage of nucleosomal DNA was tested using two different substrates: 12TP3 and 23TP2 (see Materials and Methods and references 25 and 26). In these constructs, the 12 RSS and 23 RSS are positioned approximately 30 bp and 20 bp away from the dyad, respectively. Acetylated histones were assembled on 12TP3 and 23TP2, and cleavage in the presence of different HMGB1 derivatives was assessed in comparison to cleavage of naked 12TP3 and 23TP2 DNA. In the context of a nucleosome, the single HMGB domains were less efficient at stimulating RAG1/2 cleavage than full-length HMGB1 or the derivatives containing both domains (Fig. 4). Thus, unlike cleavage of naked DNA, both HMGB domains appear to be required for efficient RAG1/2 activity in the context of chromatin.
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Nhp6A forms more stable complexes with DNA than does HMGB1. What properties of yeast Nhp6A as compared to the mammalian HMGB1 single-domain derivatives might account for the different effects of these HMGB proteins on V(D)J cleavage? Both HMGB1 and Nhp6A promote the ligation of 98-bp microcircles, with Kcircle values of 24 and 35 nM, respectively (Table 1); similar Kcircle values for HMGB1 (17 nM) and Nhp6A (38 nM) were also obtained for 75-bp microcircle formation. Moreover, both Nhp6A and HMGB1 bind to 98-bp DNA microcircles with similar apparent equilibrium dissociation constants (Table 1). However, the different behaviors of Nhp6A and HMGB1 complexes with linear DNA segments upon polyacrylamide gel electrophoresis implies that Nhp6A forms more stable DNA complexes than HMGB1 (Fig. 2B) (40).
To directly evaluate the stabilities of DNA complexes containing Nhp6A and HMGB1, complexes assembled on 98-bp microcircles were challenged with increasing amounts of sonicated salmon sperm DNA. Two Nhp6A molecules associate very tightly with the 98-bp microcircle substrates and are resistant to addition of up to 
5,000-fold mass excess of competitor DNA (Fig. 6A). By contrast the two full-length HMGB1 molecules readily dissociate from the microcircles upon addition of increasing amounts of competitor DNA.
To further quantify the differences in stability of the HMGB-DNA microcircle complexes, the kinetics of dissociation were measured after addition of 55 µg/ml competitor DNA. Complexes formed with wild-type Nhp6A on 98-bp DNA microcircles decay extremely slowly, with an estimated half-life of about 5 h (Fig. 6B; Table 1). By contrast, 50% of the complexes formed with full-length HMGB1 dissociated within 3 min, and essentially all of the complexes formed with the individual domain A' and B' peptides dissociate within 3 min (Fig. 6B; Table 1). Interestingly, HMGB1 derivatives containing both domains but lacking the acidic C-terminal tail (HMGB1 domains AB' and AB) are markedly more stable than the full-length protein, with half-lives of 26 min (Fig. 6B; Table 1). However, they remain less stable than complexes formed with wild-type Nhp6A.
Mutations in Nhp6A that decrease complex stability on microcircles enhance V(D)J cleavage.
The results described above indicate that a key difference between the properties of DNA complexes formed with Nhp6A and HMGB1 and its derivatives is their stability. This led us to consider whether Nhp6A may be assembling RAG1/2-HMGB-RSS complexes that are insufficiently dynamic to support DNA catalysis. We therefore asked if Nhp6A proteins containing mutations that destabilize their interaction with DNA were more effective at stimulating V(D)J cleavage. The basic N-terminal tail of Nhp6A, which wraps around the major groove on the opposite side of DNA from the HMGB core domain (Fig. 1D), is largely responsible for its high-affinity binding to linear DNA (29, 63). Nhp6A 
(2-12) is missing the first set of basic residues (Lys 8, Lys 9, and Arg 10; Fig. 1C). This mutant is able to bind to and promote ligation of 98-bp and 75-bp microcircles as efficiently as wild-type Nhp6A (Table 1). Nhp6A (2-12) is much more sensitive to challenge with competitor DNA than wild-type Nhp6A and exhibits a dissociation rate from 98-bp microcircles that is similar to HMGB1 domains AB and AB' (Fig. 6A and B; Table 1). Nhp6A (2-16) has the entire N-terminal basic arm region deleted. It binds poorly to linear DNA but is able to form unstable complexes on 98-bp microcircles and promote ligation of 98-bp microcircles at high protein concentrations (Table 1) (this work and reference 63). Whereas wild-type Nhp6A stimulates RAG1/2 cleavage of 12 and 23 RSS substrates poorly, reactions performed with either Nhp6A (2-12) or (2-16) generate substantial amounts of hairpins (Fig. 7A and D), while supporting complex formation at levels equivalent to wild-type Nhp6A (Fig. 7B and C). Similar results were obtained in coupled-cleavage assays; whereas Nhp6A stimulated a small amount of coupled cleavage at the 12 and 23 RSS sites, the (2-12) and (2-16) derivatives were much more effective (data not shown).
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The DNA exit wedge generated by Nhp6A Phe 48 is required for assembly of RAG1/2-HMGB-RSS complexes. Nhp6A possesses two hydrophobic wedges that insert into the base stack via the minor groove and distort the structure of the bound DNA (5, 29). Met 29 is the most important residue of the wedge, located near the center of the binding site, and Phe 48 specifies the wedge at one of the DNA exit sites (Fig. 1D). Mutants with alanine substitutions at either of these residues remain capable of forming complexes on linear DNA, but nuclear magnetic resonance analysis has revealed that both residues appear important in binding site selection (29). Phe 48 is uniquely required for recognition of the kinked DNA structure induced by cisplatin cross-linking (61). Both M29A and F48A are compromised for bending DNA, as revealed by the failure of either mutant to form 75-bp microcircles; however, M29A remains capable of forming 98-bp microcircles. Both mutants form complexes on 98-bp microcircles that are much less stable than that formed by wild-type Nhp6A but more stable than those formed by individual HMGB1 domains in the presence of competitor DNA (Fig. 6; Table 1).
The two Nhp6A wedge mutants have opposite properties with respect to the V(D)J reaction. Nhp6A F48A is unable to assemble RAG1/2-HMGB-RSS complexes (Fig. 7C) and thus also fails to promote V(D)J cleavage (Fig. 7D). By contrast, Nhp6A M29A (or Nhp6A M29D) supports RAG1/2-RSS complex formation and stimulates V(D)J cleavage relative to wild-type Nhp6A (Fig. 7C and D). A mutant containing alanine substitutions at both wedges (Nhp6A M29A F48A) behaves like Nhp6A F48A with respect to RAG1/2-HMGB-RSS complex formation and cleavage. Likewise, F48A and M29A F48A mutants failed to support coupled cleavage, but reactions performed with Nhp6A M29A or M29D exhibited enhanced cleavage at the 12 and 23 RSS sites relative to wild-type Nhp6A (data not shown). These results indicate that the Phe 48 wedge is of critical importance for initial assembly of RAG1/2-HMGB-RSS complexes. The enhanced catalytic activity of RAG1/2-HMGB-RSS complexes formed with Nhp6A M29A (and M29D) relative to wild-type Nhp6A presumably reflects the less stable binding properties exhibited by the mutant (Fig. 6A and B; Table 1).
Requirement for Phe 38 of HMGB1 domain A for RAG1/2-RSS complex formation. Phe 38 in HMGB1 domain A corresponds to Phe 48 of Nhp6A. We therefore asked whether Phe 38 in the domain A peptide was similarly required for the assembly of RAG1/2-RSS complexes. Domain A peptide containing the F38A mutation failed to promote ligation of 98-bp microcircles, although it still was able to form unstable complexes with preformed 98-bp microcircles (Table 1). Similarly, domain A F38A does not promote assembly of RAG1/2 complexes on the 23 RSS, and therefore, as expected, no stimulation of cleavage by the mutant HMGB1 derivative is detected (Fig. 8).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relationship between the DNA binding properties of resected derivatives of mammalian HMGB1 and their support of RAG1/2-RSS complex assembly and cleavage activity. (i) HMGB1 domain A versus domain B. The rat HMGB1 DNA binding domains (A and B) are 29% identical and 50% similar to each other (Fig. 1C). Our recombinant versions of domains A' and B' containing their C-terminal basic ends function very similarly with respect to their abilities to form as well as to selectively bind to DNA microcircles. The similarity in DNA bending efficiencies between domains A' and B' observed here contrasts with some other studies where domain B, with its two intercalating wedges, has been found to exhibit greater bending activity (46, 52, 64). However, we note that the recombinant form of domain B used here lacks the lysine-rich linker region (amino acids 85 to 88; TKKK) separating domains A and B, which has been reported to enhance DNA binding (17, 46, 47). Our minimal-domain peptides that lack the basic residues at their C-terminal ends show pronounced differences in bending and binding to microcircular DNA; domain A requires less protein than domain B for equivalent activity. We conclude that both HMGB1 domains are effective at bending DNA but that flanking basic residues enhance their activities, particularly for the B domain.
The DNA binding and bending properties of the isolated domains largely parallel their activities in the V(D)J reaction on naked DNA. Whereas each of the isolated domain constructs are able to efficiently promote RAG1/2-RSS assembly and V(D)J cleavage, the B'- and B-domain peptides require more protein than the respective A'- and A-domain peptides for optimal activity. However, the ability to bend DNA, as evaluated by microcircle formation, is not the sole determinant of HMGB function in the V(D)J reaction, since the domain A' peptide behaved nearly indistinguishably from the didomain derivatives on naked DNA, even though 10- or 100-fold more domain A' was required to generate equivalent numbers of microcircles compared to full-length HMGB1 or domains AB/AB', respectively. As elaborated further below, the differences that we observe with the minimal domains are consistent with the view that domain A of HMGB1 is more active than domain B in interacting with prebent and structured DNA (19, 22, 52, 59) and that a localized DNA disruption exists within the RAG1/2-HMGB-RSS assembly (4, 10, 11, 42).
(ii) The C-terminal acidic tail. The C-terminal acidic tail has generally been found to inhibit binding of HMGB1 derivatives to naked DNA, and our results are consistent with this view (22, 27, 48). Nevertheless, full-length HMGB1 appears no less efficient at promoting RAG1/2 interactions on RSS DNA than domain A or didomain derivatives lacking the C-terminal tail. As others also have noted, the single domain B" peptide containing the acidic region exhibits very poor DNA binding and bending activities, and consequently it appears essentially inactive in the V(D)J reaction. Recent nuclear magnetic resonance and cross-linking experiments have provided evidence that the acidic region can specifically interact with residues within both domains and the connecting basic linker segment, which presumably inhibits DNA binding (22, 24).
The acidic region in the context of the didomain structure has been reported to facilitate HMGB1 interactions with nucleosomes, possibly by interacting with the histone H3 N terminus (56). Bonaldi et al. have reported that the presence of the acidic region on HMGB1 enhances nucleosome sliding by the ACF (ISWI) remodeling complex (8). We find that full-length HMGB1 is somewhat more efficient in promoting RSS cleavage by RAG1/2 on nucleosomal substrates than the didomain derivatives without the acidic region, suggesting that the acidic region could have a modest beneficial role in V(D)J recombination in vivo.
Nhp6A mutants reveal parameters important for assembly of catalytically competent RAG1/2-RSS complexes. The sequence and structure of the HMGB domain of Nhp6A are more closely related to HMGB1 domain B (48% amino acid residue identity, 1.2-Å root mean square deviation between peptide backbones) than domain A (26% amino acid residue identity, 2.2-Å root mean square deviation between peptide backbones) (Fig. 1C) (5, 29). Nhp6A binds DNA much more stably than any of the HMGB1 derivatives, primarily because of its N-terminal basic arm. We find that the greater affinity for DNA does not improve RAG1/2-RSS complex formation. Nhp6A promotes efficient RAG1/2-RSS complex formation at protein levels that are equivalent to HMGB1 domain A' or the didomain constructs. However, the stable DNA binding by Nhp6A strongly inhibits DNA cleavage by the RAG1/2-Nhp6A-RSS complexes. Mutations within the N-terminal tail or within the HMGB core that destabilize binding and thus lead to Nhp6A proteins whose DNA binding properties more closely mimic those of the HMGB1 derivatives result in corresponding increases in V(D)J cleavage. These findings suggest that a dynamic association of the HMGB protein with the RAG1/2-RSS complex is critical for the DNA cleavage steps. The HMGB protein is not released from the complex, however, since HMGB1 has been shown to directly enhance hairpin formation in experiments utilizing prenicked substrates and to remain with the complex after the hairpin has been formed (20, 33, 34, 49, 50, 57).
Nhp6A contains two hydrophobic wedges at the DNA binding surface, but only one, Phe 48, is critical for stimulating RAG1/2-RSS assembly. This residue corresponds to Phe 38 in domain A, which we also show to be essential for its ability to promote RAG1/2-RSS assembly, and to Ile 34 in domain B (Fig. 1D). The importance of Phe 48 and the dispensability of the Met 29 wedge parallel the roles of these two residues in selective binding by Nhp6A to cisplatin adducts on DNA (61). Consistent with its moderately poorer activity in the V(D)J reaction, HMGB1 domain B has been found to be less selective for binding to cisplatin adducts than domain A, in part because of the isoleucine in place of a phenylalanine at the DNA exit wedge (19, 22). Cisplatin forms an intrastrand cross-link at the N7 position of adjacent guanines, generating a kink in the duplex DNA that compresses the major groove (51). An X-ray structure of mammalian HMGB1 domain A bound to a DNA duplex containing a cisplatin cross-link revealed that the Phe 38 intercalates between the cross-linked guanines on the minor groove side (Fig. 1D) (19, 38).
Aidinis et al. have provided evidence for bending of the RSS sites by RAG1/2 by circular permutation analysis of the electrophoretic migrations of RAG1/2-12 or 23 RSS complexes, even in the absence of HMGB1 (4). This finding, combined with the importance of the phenylalanine wedge, suggests that the HMGB protein may be recognizing a RAG1/2-induced DNA distortion between the nonamer and heptamer regions. Binding of the HMGB proteins may then stabilize the bent structure, thus enhancing complex formation. Consistent with this model, both protein-DNA cross-linking and ethylation interference experiments have suggested that HMGB1 is located in the intervening DNA segment. Ethylation interference experiments point to the HMGB protein contacting DNA near the 5' end of the nonamer on the opposite side of the duplex from the bound RAG1/2 (33, 49). There are also several lines of evidence suggesting that unpairing of DNA strands, particularly over the heptamer region, may be important for the DNA cleavage/hairpin formation steps (10 and reviewed in reference 14). HMGB proteins induce considerable untwisting of DNA along with bending, which may further contribute to their stimulation of RAG1/2 function.
Comparison of HMGB1 stimulation of RAG1/2 and other DNA binding proteins. The DNA binding domain of RAG1 has been proposed to contain a helix-turn-helix motif that is similar to the Tc1/mariner family of transposases (6). Interestingly, a member of the Tc1/mariner family of transposons, Sleeping Beauty, has also been shown to require HMGB1 for transposition in human cells (66). HMGB1 also facilitates binding of a number of transcription factors with structurally diverse DNA binding domains (2, 54). As is the case for RAG1/2 binding, a single domain from HMGB1 has been found to be sufficient in stimulating DNA binding of some of these regulatory proteins, including the Oct1/2 and HOXD9 homeodomains and Rta (31, 32, 65, 68). However, in at least one case, the cooperative binding of the basic leucine zipper protein Zebra to adjacent sites within the regulatory region of an Epstein-Barr virus promoter, both linked domains of HMGB1 are required (12). In some situations, there is evidence that HMGB1 interacts in solution with its binding partner, implying that direct protein-protein interactions, in addition to effects on DNA architecture by HMGB1, appear to contribute to binding cooperativity (reviewed in (2, 54). On the other hand, stable ternary complexes containing HMGB1 and DNA have not been observed upon polyacrylamide gel electrophoresis in several systems. Therefore, the HMGB protein appears to function in some contexts only transiently to chaperone the transcription factor to its DNA binding site.
Aidinis et al. reported that RAG1/2 and HMGB1 interact in solution in experiments employing immobilized RAG1/2 or HMGB1 (4). Both domains A and B were required in order to observe this interaction, leading the authors to propose that both domains are required for HMGB1 stimulation of RAG1/2 activity. Our finding that either isolated HMGB1 domain or unstable binding mutants of yeast Nhp6A are effective in promoting the assembly of catalytically competent RAG1/2-HMGB-RSS complexes suggests that a specific RAG1/2-HMGB interaction may be secondary to an effect of HMGB on DNA architecture. Only nine solvent-exposed residues, which have similar chemical characteristics but are not directly involved in DNA binding, are common between the three HMGB domains (Fig. 1C).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: Cancer Research Center, Boston University School of Medicine, 715 Albany St., Boston, MA 02118.
Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142.
Present address: Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, South Korea.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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