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• W2156953452 abstract "RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the “HMG-box” family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants. RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the “HMG-box” family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants. During lymphocyte development, antigen receptor genes undergo a series of DNA rearrangements to generate functional exons encoding the antigen binding domains of these receptors (1Bassing C.H. Swat W. Alt F.W. Cell. 2002; 109: S45-S55Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). This rearrangement process, termed V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called recombination-activating gene (RAG) 1The abbreviations used are: RAG, recombination-activating gene; DSB, double-strand break; RSS, recombination signal sequence; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; NHEJ, non-homologous end-joining; HMG, high mobility group; MBP, maltose-binding protein; IMAC, immobilized metal affinity chromatography; EMSA, electrophoretic mobility shift assay; LAR-PCR, ligation-assisted recombination polymerase chain reaction; IDA, iminodiacetate; IEC, ion exchange chromatography; WT, wild type; SB, sleeping beauty; PC, paired complex. 1The abbreviations used are: RAG, recombination-activating gene; DSB, double-strand break; RSS, recombination signal sequence; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; NHEJ, non-homologous end-joining; HMG, high mobility group; MBP, maltose-binding protein; IMAC, immobilized metal affinity chromatography; EMSA, electrophoretic mobility shift assay; LAR-PCR, ligation-assisted recombination polymerase chain reaction; IDA, iminodiacetate; IEC, ion exchange chromatography; WT, wild type; SB, sleeping beauty; PC, paired complex.-1 and RAG-2, bring two gene segments into close proximity and then introduce a DNA double-strand break (DSB) at the end of each coding segment. Adjacent to each coding segment lies a recombination signal sequence (RSS) that serves as the binding site for the RAG-1/2 protein complex (hereafter termed the “RAG complex”) and directs the location of cleavage. The RSS contains a conserved heptamer and nonamer motif separated by either 12 or 23 bp of relatively nonconserved sequence (12-RSS and 23-RSS, respectively). Productive exon assembly is promoted by the 12/23 rule, a restriction that limits rearrangement to RSSs whose spacing between the heptamer and nonamer is different. RAG-mediated cleavage of RSS pairs produces four DNA ends: two blunt, 5′-phosphorylated signal ends and two coding ends terminating in DNA hairpin structures (2Roth D.B. Zhu C. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10788-10792Crossref PubMed Scopus (155) Google Scholar, 3Roth D.B. Menetski J.P. Nakajima P.B. Bosma M.J. Gellert M. Cell. 1992; 70: 983-991Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 4Schlissel M. Constantinescu A. Morrow T. Baxter M. Peng A. Genes Dev. 1993; 7: 2520-2532Crossref PubMed Scopus (337) Google Scholar). These reaction products originate from a two-step cleavage reaction in which the RSS is first nicked at its 5′-end, and then the resulting 3′-OH is covalently linked to the bottom strand by direct trans-esterification (5McBlane J.F. van Gent D.C. Ramsden D.A. Romeo C. Cuomo C.A. Gellert M. Oettinger M.A. Cell. 1995; 83: 387-395Abstract Full Text PDF PubMed Scopus (585) Google Scholar, 6van Gent D.C. Mizuuchi K. Gellert M. Science. 1996; 271: 1592-1594Crossref PubMed Scopus (240) Google Scholar). After DNA cleavage, signal ends are generally ligated together to form precise signal joints, but coding ends, being sealed as DNA hairpins, are first resolved and then processed and joined to create coding joints in which nucleotides are frequently gained or lost at the junction. DNA hairpin opening is most likely catalyzed by a complex containing Artemis and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) (7Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). Signal and coding joint formation is mediated by ubiquitously expressed proteins that comprise the non-homologous end-joining pathway (NHEJ) of DNA double-strand break repair, including Ku70, Ku80, XRCC4, and DNA ligase IV (1Bassing C.H. Swat W. Alt F.W. Cell. 2002; 109: S45-S55Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar).Early studies of RAG protein biochemistry demonstrated that both proteins were necessary and sufficient to cleave a DNA substrate containing a single RSS in vitro (5McBlane J.F. van Gent D.C. Ramsden D.A. Romeo C. Cuomo C.A. Gellert M. Oettinger M.A. Cell. 1995; 83: 387-395Abstract Full Text PDF PubMed Scopus (585) Google Scholar). Subsequent studies revealed that RAG complex binding to isolated recombination signals (especially a 23-RSS), and RAG-mediated synapsis and cleavage of RSS pairs according to the 12/23 rule are promoted by “architectural” DNA-binding proteins of the HMG-box family of high mobility group (HMG) proteins (8van Gent D.C. Hiom K. Paull T.T. Gellert M. EMBO J. 1997; 16: 2665-2670Crossref PubMed Scopus (214) Google Scholar). In this respect, the RAG complex is part of a growing group of recombinases and transcription factors that are known to associate with, and have their activity augmented by, HMG-box proteins (9Bustin M. Mol. Cell Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (746) Google Scholar, 10Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). How these proteins stimulate the DNA binding and cleavage activity of the RAG complex remains poorly understood. Here, we investigate the mechanisms by which a prototypical HMG-box protein, HMGB1, facilitates RAG-RSS complex assembly and activity.Mammalian members of the HMG-box family (including HMGB1 and HMGB2) contain tandem homologous DNA binding domains, termed HMG-box A and B, each of which consist of about 80 amino acids folded into three α-helices that adopt a characteristic L-shaped structure. The two HMG-box domains are followed by a short linker rich in basic amino acid residues and a C-terminal acidic tail comprised of about 30 consecutive aspartate and glutamate residues (10Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Structural and biochemical studies of the individual HMG-box domains suggest both interact with the minor groove of DNA, but display different DNA binding properties: box A exhibits a preference for binding distorted DNA structures such as four-way junctions (11Webb M. Thomas J.O. J. Mol. Biol. 1999; 294: 373-387Crossref PubMed Scopus (82) Google Scholar) and DNA interstrand cross-links (12Kasparkova J. Delalande O. Stros M. Elizondo-Riojas M.A. Vojtiskova M. Kozelka J. Brabec V. Biochemistry. 2003; 42: 1234-1244Crossref PubMed Scopus (39) Google Scholar), whereas box B does not selectively bind such DNA structures, but can itself introduce a severe bend into linear DNA when flanking sequences are present (13Teo S.H. Grasser K.D. Thomas J.O. Eur. J. Biochem. 1995; 230: 943-950Crossref PubMed Scopus (98) Google Scholar, 14Stros M. J. Biol. Chem. 1998; 273: 10355-10361Abstract Full Text Full Text PDF PubMed Google Scholar). (The A domain lacks this ability.) The acidic tail of HMG-box proteins has been shown to mediate interactions with the HMG-boxes (15Knapp S. Muller S. Digilio G. Bonaldi T. Bianchi M.E. Musco G. Biochemistry. 2004; 43: 11992-11997Crossref PubMed Scopus (80) Google Scholar), as well as with core histones, thereby promoting association of the HMG-boxes with nucleosome linker DNA (16Ueda T. Chou H. Kawase T. Shirakawa H. Yoshida M. Biochemistry. 2004; 43: 9901-9908Crossref PubMed Scopus (69) Google Scholar). In addition, the acidic tail plays a role in stimulating nucleosome sliding by chromatin remodeling factors (17Bonaldi T. Langst G. Strohner R. Becker P.B. Bianchi M.E. EMBO J. 2002; 21: 6865-6873Crossref PubMed Scopus (187) Google Scholar). The distinct functional properties of the HMG-boxes and the acidic tail cause us to speculate that these regions may play separable roles in the assembly and activity of RAG-RSS complexes.To explore this possibility, we prepared an extensive panel of truncated and mutant HMGB1 proteins and systematically tested their ability to promote RAG complex binding and cleavage of single and paired RSS substrates using a combination of mobility shift and in-gel cleavage assays. We find that, like full-length HMGB1, individual HMG-box domains supershift RAG-RSS complexes. Forms of full-length HMGB1 bearing alanine substitutions at residues thought to mediate important protein-DNA contacts in either or both HMG-boxes also supershift RAG-RSS complexes. Interestingly, however, despite being incorporated into RAG-RSS complexes, individual HMG-box domain proteins and full-length HMGB1 bearing mutations in box B fail to stimulate RAG-mediated DNA cleavage of single RSS substrates. Mutations in box A reduce, but do not eliminate the ability of full-length HMGB1 to enhance RAG-mediated cleavage. However, the box A mutant is less effective at promoting site-specific cleavage of a 23-RSS, and stimulates 12/23-regulated cleavage poorly. Removal of the basic linker and/or the acidic tail from HMGB1 do not appreciably reduce its ability to stimulate RAG-mediated cleavage, but increases the nonspecific DNA binding activity of HMGB1, and enables mutant forms of HMGB1 to promote assembly of RAG synaptic complexes, which otherwise is impaired when these mutants contain the acidic tail. Interestingly, a tailless form of HMGB1 in which the positions of HMG-box A and B are reversed stimulates RAG-mediated synapsis and cleavage only slightly less effectively than its normally oriented counterpart. Taken together, these data argue that box B, likely by promoting DNA bending through interactions with RAG-1, plays a critical role in promoting RAG-mediated cleavage, but is not sufficient by itself to correctly position the recombinase active site in a 23-RSS, which requires the activity of a functional tethered box A. Moreover, the acidic tail may help enforce the 12/23 rule by reducing nonspecific protein-DNA interactions.EXPERIMENTAL PROCEDURESExpression Vectors—Eukaryotic expression constructs encoding core RAG-1 or core RAG-2, each fused at the amino terminus to MBP without additional sequence tags (pcMR1 and pcMR2, respectively), have been described previously (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar). The prokaryotic expression construct pET11d-hHMGB1, encoding full-length human HMGB1 tagged at the amino terminus with polyhistidine, has been described elsewhere (19Ge H. Roeder R.G. J. Biol. Chem. 1994; 269: 17136-17140Abstract Full Text PDF PubMed Google Scholar). Derivatives of this construct encoding forms of hHMGB1 that are truncated or contain alanine substitutions, as depicted in Fig. 1, were generated as described under supplemental materials.Protein Expression and Purification—RAG-1 and RAG-2 fusion proteins were coexpressed in 293 cells and purified as described elsewhere (20Swanson P.C. Volkmer D. Wang L. J. Biol. Chem. 2004; 279: 4034-4044Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Wild-type, truncated, and mutant forms of HMGB1 were expressed in Escherichia coli strain BL21(DE3)pLysS and initially purified by immobilized metal affinity chromatography (IMAC) using iminodiacetate (IDA)-coupled Sepharose charged with Ni2+. Subsequent purification steps involved ion exchange chromatography (IEC) using strongly acidic (High S), weakly acidic (CM), or strongly basic (High Q) ion exchange supports as schematically diagrammed in Fig. 1. The purification procedures are described in detail under supplemental materials. We consistently observed lower overall yields of HMGB1 bearing alanine substitutions (either HMG-box, but particularly mtA), as these forms of HMGB1 are prone to degradation (not shown).Oligonucleotide Binding Assays—Intact 12- and 23-RSS substrates were assembled and purified as described previously (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar, 21Li W. Swanson P. Desiderio S. Mol. Cell. Biol. 1997; 17: 6932-6939Crossref PubMed Scopus (24) Google Scholar). Electrophoretic mobility shift assays (EMSA) were performed under the same conditions as described previously (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar, 22Swanson P.C. Desiderio S. Immunity. 1998; 9: 115-125Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The amounts of HMGB1 used in analytical EMSAs are noted in the figures or figure legends. Protein-DNA complexes were visualized from dried gels by autoradiography using a Molecular Dynamics Storm 860 Gel and Blot Imaging System.In-gel Cleavage Assays—Preparative scale binding reactions were assembled in the presence of Ca2+, fractionated by EMSA, and protein-DNA complexes assayed for cleavage activity using a previously described in-gel cleavage assay (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar, 23Swanson P.C. Mol. Cell. Biol. 2001; 21: 449-458Crossref PubMed Scopus (51) Google Scholar).RESULTSPreparation of Wild-type, Truncated, and Mutant Forms of HMGB1—In a previous study, the activities of HMGB1 and HMGB2 were directly compared in RAG-RSS binding and cleavage assays (24Swanson P.C. Mol. Cell. Biol. 2002; 22: 1340-1351Crossref PubMed Scopus (64) Google Scholar). In that study, HMGB1 and HMGB2 were found to comparably supershift RAG-RSS complexes containing either a 12- or 23-RSS, and stimulate RAG-mediated cleavage of an isolated 23-RSS, but not a 12-RSS, using an in-gel cleavage assay. In a subsequent study, HMGB1 was found to be required for RAG-mediated synapsis and cleavage of intact RSS pairs according to the 12/23 rule, and 12/23-regulated cleavage of nicked RSS pairs, despite not being rigorously required for synapsis of nicked substrates (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar). In addition, HMGB1 was found to promote formation of RAG complexes with cleaved signal ends, but once formed, the RAG proteins did not require HMGB1 to mediate transposition of signal ends into a plasmid target substrate (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar). To extend these studies, we wished to identify the structural features of HMGB1 responsible for stimulating RAG-mediated binding and cleavage of an isolated 23-RSS, and synapsis and 12/23-regulated cleavage of intact RSS pairs.Sequence, structural, and biochemical characterization of HMGB1 and related HMG-box proteins suggest that HMG box A, box B, the basic linker, and the acidic tail of HMGB1 are encompassed within the amino acid residues shown in Fig. 1A (10Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). To systematically test how the various regions of HMGB1 affect RAG binding and cleavage activity, we prepared an extensive panel of bacterially expressed truncated and mutant forms of HMGB1 (Fig. 1A, see supplemental materials). To disrupt the DNA binding activity of HMGB1, we elected to replace ten consecutive residues with alanine at comparable positions in helix 1 of either or both HMG-boxes, starting at residue 18 of box A and residue 102 of box B, using ligation-assisted recombination PCR mutagenesis (LAR-PCR, see supplemental materials and supplemental Fig. 1). These residues were targeted for mutagenesis based on sequence and structural analysis indicating that they comprise part of a “hydrophobic wedge” in their respective HMG-box domains that mediates contact with the minor groove of DNA (10Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Phenylalanine 103, for example, is thought to intercalate between DNA base pairs, promoting DNA bending; mutation of this residue impairs the DNA supercoiling activity of box B (25Stros M. Muselikova E. J. Biol. Chem. 2000; 275: 35699-35707Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Mutagenesis also replaces a highly conserved arginine residue present in both HMG-boxes (Arg24 in box A, and Arg110 in box B); substitution of this residue in either HMG-box reduces the DNA binding activity of the domain (25Stros M. Muselikova E. J. Biol. Chem. 2000; 275: 35699-35707Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Mitsouras K. Wong B. Arayata C. Johnson R.C. Carey M. Mol. Cell Biol. 2002; 22: 4390-4401Crossref PubMed Scopus (77) Google Scholar). To test whether the orientation of the two HMG-box domains relative to one another is an important determinant for the ability of HMGB1 to stimulate RAG complex binding and cleavage activity, we also prepared a version of HMGB1 in which the positions of the two HMG-box domains are inverted or “shuffled.” Purifying the various HMGB1 proteins required different strategies, which are schematically diagrammed in Fig. 1B and described in detail under supplemental materials. Using these purification strategies, we obtained highly purified truncated and mutant HMGB1 proteins estimated to be between 90 and 95% pure (Fig. 1C).Truncated and Mutant Forms of HMGB1 Differentially Supershift RAG-RSS Complexes—HMGB1 and HMGB2 are known from previous studies to become incorporated into RAG-RSS complexes, causing the protein-DNA complex to migrate more slowly in an EMSA (24Swanson P.C. Mol. Cell. Biol. 2002; 22: 1340-1351Crossref PubMed Scopus (64) Google Scholar). To compare the ability of full-length, truncated and mutant forms of HMGB1 to supershift RAG-RSS complexes, purified RAG proteins were incubated with a radiolabeled 23-RSS substrate in the presence of increasing concentrations of the various forms of HMGB1, and protein-DNA complex formation analyzed by EMSA (Fig. 2). All forms of HMGB1 tested, except B′, were found to supershift RAG-RSS complexes in a concentration-dependent manner. Based on these binding titrations, the concentration of each form of HMGB1 (except B′) needed to visibly supershift the RAG-RSS complexes was used in an EMSA to compare the mobility of supershifted protein-DNA complexes side-by-side, both in the absence and presence of the RAG proteins (Fig. 3).Fig. 2Truncated and mutant forms of HMGB1 exhibit differential capacity to supershift RAG-RSS complexes. Coexpressed MBP-RAG-1 and MBP-RAG-2 (RAG-1/2) was incubated with a radiolabeled intact 23-RSS substrate in the absence or presence of increasing amounts of the following forms of HMGB1 (as indicated above the gel): wild-type or tailless (A), tailless mtA or tailless mtB (B), basic (C), mtB (D), box A or shuffled (E), mtAB or mtA (F), box B′ (G), or box B (H). Protein-DNA complexes were fractionated by EMSA, and visualized from dried gels using a phosphorimager.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Comparative binding of various HMGB1 proteins to a 23-RSS in the absence or presence of the RAG complex. Purified forms of HMGB1 were incubated with a radiolabeled intact 23-RSS substrate in the absence or presence of the RAG complex (as indicated above the gels), and protein-DNA complex formation was analyzed using EMSA. A, EMSA of individual HMG-boxes and full-length forms of HMGB1. B, EMSA of basic, tailless, and shuffled forms of HMGB1. Based on the titrations performed in Fig. 2, the following amounts of each form of HMGB1 were used in the binding reaction: 150 ng of wild type, mtA, mtB, or mtAB; 50 ng of box A, basic, tailless, tailless mtA, tailless mtB, or shuffled. Because B′ failed to supershift RAG-RSS complexes at the concentrations tested (see Fig. 2), we used 150 ng in this assay for comparison to other forms of HMGB1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the absence of HMGB1, the RAG proteins form two distinct protein-DNA complexes when assembled on substrates containing a single RSS. We have previously shown that these two RAG-RSS complexes, called SC1 and SC2, both contain a RAG-1 dimer, but differ in the stoichiometry of RAG-2, with SC1 containing monomeric RAG-2 and SC2 containing two RAG-2 molecules (18Swanson P.C. Mol. Cell. Biol. 2002; 22: 7790-7801Crossref PubMed Scopus (69) Google Scholar). Both RAG-RSS complexes are supershifted in the presence WT HMGB1 and individual HMGB boxes A and B, but not box B′, presumably because the negative charge associated with the acidic tail interferes with DNA binding (Figs. 2 and 3). Mixing HMG box A with either form of box B promotes formation of supershifted RAG-RSS complexes that comigrate with those formed in the presence of box A alone (Fig. 3). HMGB1 bearing mutations in box B supershift RAG-RSS complexes comparably to WT HMGB1, regardless of the presence of the acidic tail (Fig. 2). However, slightly more (∼2-fold) mtA HMGB1 is required to reach a comparable shift in the mobility of the RAG-RSS complexes relative to WT or mtB HMGB1, although this difference is diminished if the acidic tail is removed (Fig. 2). Interestingly, mtAB HMGB1 exhibits a subtle and selective defect in its ability to supershift SC1, but supershifts SC2 comparably to mtA and mtB HMGB1 (Fig. 3). The observation that both mtA and mtB HMGB1 bind an isolated RSS and also supershift the SC1 and SC2 RAG complexes suggests that the introduced mutations do not severely disrupt the global folding of the protein, although some loss of α-helical content is observed in mtA and mtB HMGB1 compared with WT HMGB1, as indicated by circular dichroism spectroscopy (data not shown). Consistent with previous observations (27Lee K.B. Thomas J.O. J. Mol. Biol. 2000; 304: 135-149Crossref PubMed Scopus (76) Google Scholar), tailless forms of HMGB1 exhibit greater DNA binding activity than full-length HMGB1, and are prone to forming higher order oligomeric complexes with DNA as a function of protein concentration, both in the absence and presence of the RAG proteins (Figs. 2 and 3). Notably, about 5-10-fold more full-length HMGB1 is required to visibly supershift the RAG-RSS complexes compared with its tailless counterparts (Fig. 2). This effect is largely attributed to the presence of the acidic tail, as similar concentrations of basic and tailless HMGB1 are required to supershift the RAG-RSS complexes (Fig. 2). Perhaps surprisingly, “shuffled” HMGB1 also supershifts SC1 and SC2, exhibiting a concentration dependence similar to that observed for tailless HMGB1.Separable Roles for the HMG-box Domains in Facilitating RAG-mediated Cleavage of an Intact 23-RSS—One limitation of the mobility shift experiments described in the previous section is that they do not provide information about the functional significance of HMGB1 protein association with the RAG-RSS complex. To address this issue, we evaluated the ability of the various HMGB1 proteins to stimulate RAG-mediated cleavage of an intact 23-RSS within the context of the supershifted SC1 RAG-RSS complex using a previously described in-gel cleavage assay that enables a direct comparison of cleavage activity between multiple, discrete RAG-RSS complexes fractionated on a single nondenaturing polyacrylamide gel (23Swanson P.C. Mol. Cell. Biol. 2001; 21: 449-458Crossref PubMed Scopus (51) Google Scholar) (Fig. 4). As a negative control, the activity of a catalytically defective RAG complex (RAG-1 D600A) was analyzed in parallel with protein-DNA complexes assembled with WT RAG proteins. In these assays, we intentionally used the lowest concentration of the various forms of HMGB1 that yield a detectable supershift of the RAG complex to reduce the possibility of nonspecific binding by the HMGB1 proteins, while concomitantly retaining the ability to unambiguously determine whether the ability to supershift the RAG-RSS complex is the sole determinant for altering its activity.Fig. 4Stimulation of RAG-mediated 23-RSS cleavage by HMGB1 is primarily mediated by box B, but requires a tethered box A. A, in-gel cleavage assay of RAG-RSS complexes supershifted by different forms of HMGB1. A wild-type or inactive RAG complex (WT cMR1/cMR2 and D600A cMR1/cMR2, respectively) was incubated with radiolabeled intact 23-RSS either alone or with various forms of HMGB1 (indicated above the gel) in preparative binding reactions containing Ca2+ and protein-DNA complexes were fractionated by EMSA. After electrophoresis, gels were submerged in buffer containing Mg2+ for 1 h at 37 °C to initiate cleavage and then the DNA was transferred to DEAE cellulose paper. Reaction products recovered from complexes of interest (identified by autoradiography; see exposure at the top) were then analyzed on a 15% polyacrylamide sequencing gel (bottom). Positions of nicked, hairpin, and aberrantly nicked products are indicated at the left. Note that samples from lanes 1-9 and 10-15 were obtained from different in-gel cleavage assays (given space limitations for the EMSA), but all reaction products were fractionated on the same sequencing gel. B, quantification of reaction products. The abundance of aberrant nicks and hairpins (top bar graph) or correctly positioned nicks (bottom bar graph) are shown for each lane depicted in A and presented in the same order. The data are representative of independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Consistent with previous results (24Swanson P.C. Mol. Cell. Biol. 2002; 22: 1340-1351Crossref PubMed Scopus (64) Google Scholar), a RAG-RSS SC1 complex supershifted by wild-type, full-length HMGB1 catalyzes more site-specific nicking and hairpin formation, while exhibiting less aberrant nicking in the spacer region of the 23-RSS, than an unshifted SC1 complex. Despite its ability to supershift the SC1 complex, box A alone does not appreciably stimulate nicking or hairpin formation when present in this complex. Because box B′ containing the acidic tail does not supershift the SC1 complex, it is perhaps not surprising that the complex formed in the presence of this form of HMGB1 does not stimulate cleavage. Interestingly, the supershifted SC1 complex formed in the presence of both box A and box B′ proteins exhibits no more activity than the SC1 complex supershifted by box A alone. This observation holds true regardless of whether or not the acidic tail is present on box B (data not shown).When incorporated into the SC1 complex, mtA HMGB1 domain stimulates nicking and hairpin formation, albeit to lower levels than WT HMGB1. However, in contrast to WT HMGB1, there is a small increase in the abundance of the aberrantly nicked product. Perhaps more importantly, and in marked contrast with the mtA HMGB1, an SC1 complex supershifted with mtB HMGB1 is no more active than an unshifted SC1 complex in the catalysis of appropriately sited nicks and DNA hairpins. However, similarly to mtA HMGB1, a modest increase in aberrant nicks is reproducibly observed in this complex. This effect is also observed when both HMG-box domains" @default.
• W2156953452 created "2016-06-24" @default.
• W2156953452 creator A5001129135 @default.
• W2156953452 creator A5021876418 @default.
• W2156953452 creator A5025226546 @default.
• W2156953452 date "2005-09-01" @default.
• W2156953452 modified "2023-09-27" @default.
• W2156953452 title "Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro" @default.
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