SMART MODE:

NORMAL GENOMIC

• Nakamura Y, Yoshioka K, Shirakawa H, Yoshida M
• HMG box A in HMG2 protein functions as a mediator of DNA structural alteration together with box B.
• J Biochem (Tokyo). 2001; 129: 643-51
• Display abstract

• Nonhistone protein HMG2, like HMG1, binds with B-DNA in a sequence-nonspecific manner and causes structural alterations in DNA such as bending, kinking and unwinding. Here, we studied the functions of HMG2 domains in the DNA structural alteration and modulation by using various HMG2 peptides, and we demonstrated several new findings. The HMG box itself as a DNA-binding motif may have the basic function of inducing curvature, resulting in the apparent DNA bending in the DNA cyclization assay, but not of abruptly kinking DNA. The DNA-binding activity of HMG box B, which is enhanced by the presence of box A, together with the flanking regions of box B, causes DNA bending accompanying the kinking of the DNA main chain. The DNA unwinding accompanied by DNA kinking diminishes cruciform structures in supercoiled DNA. Analysis using mutant peptides for box A confirmed that box A in HMG2 functions as a mediator of DNA structural alteration together with box B. The present studies on the functional properties of the respective regions of HMG2 may help to elucidate the protein function.

• Thomas JO
• HMG1 and 2: architectural DNA-binding proteins.
• Biochem Soc Trans. 2001; 29: 395-401
• Display abstract

• HMG1 and 2 (high mobility group proteins 1 and 2; renamed HMGB1 and 2) contain two DNA-binding HMG-box domains (A and B) and a long acidic C-terminal domain. They bind DNA without sequence specificity, but have a high affinity for bent or distorted DNA, and bend linear DNA. The individual A and B boxes (which, although broadly similar, show both structural and functional differences) exhibit many of the structure-specific properties of the whole protein. The acidic tail modulates the affinity of the tandem HMG boxes in HMG1 and 2 for a variety of DNA targets, including four-way junctions, but not distorted DNA minicircles, to which the proteins bind with very high affinity. HMG1 and 2 appear to play important architectural roles in the assembly of nucleoprotein complexes in a variety of biological processes, for example V(D)J recombination, the initiation of transcription, and DNA repair.

• Thomas JO, Travers AA
• HMG1 and 2, and related 'architectural' DNA-binding proteins.
• Trends Biochem Sci. 2001; 26: 167-74
• Display abstract

• The HMG-box proteins, one of the three classes of high mobility group (HMG) chromosomal proteins, bend DNA and bind preferentially to distorted DNA structures. The proteins appear to act primarily as architectural facilitators in the assembly of nucleoprotein complexes; for example, in effecting recombination and in the initiation of transcription. HMG-box proteins might be targeted to particular DNA sites in chromatin by either protein-protein interactions or recognition of specific DNA structures.

• Weiss MA
• Floppy SOX: mutual induced fit in hmg (high-mobility group) box-DNA recognition.
• Mol Endocrinol. 2001; 15: 353-62
• Display abstract

• The high-mobility group (HMG) box defines a DNA-bending motif of broad interest in relation to human development and disease. Major and minor wings of an L-shaped structure provide a template for DNA bending. As in the TATA-binding protein and a diverse family of factors, insertion of one or more side chains between base pairs induces a DNA kink. The HMG box binds in the DNA minor groove and may be specific for DNA sequence or distorted DNA architecture. Whereas the angular structures of non-sequence-specific domains are well ordered, free SRY and related autosomal SOX domains are in part disordered. Observations suggesting that the minor wing lacks a fixed tertiary structure motivate the hypothesis that DNA bending and stabilization of protein structure define a coupled process. We further propose that mutual induced fit in SOX-DNA recognition underlies the sequence dependence of DNA bending and enables the induction of promoter-specific architectures.

• Stros M
• Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA.
• Biochemistry. 2001; 40: 4769-79
• Display abstract

• High mobility group (HMG) 1 protein and its two homologous DNA-binding domains, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of topoisomerase I, as well as recognize differently bent and distorted DNA structures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (85)TKKKFKD(91) sequence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DNA. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modified DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180--2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cisplatin-modified DNA, inhibits the ability of the B domain to bend (ligase-mediated circularization) or supercoil DNA, and results in a loss of the preferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of the reported activities of the B domain are enhanced when the B domain is covalently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and other activities.

• Papoulas O, Daubresse G, Armstrong JA, Jin J, Scott MP, Tamkun JW
• The HMG-domain protein BAP111 is important for the function of the BRM chromatin-remodeling complex in vivo.
• Proc Natl Acad Sci U S A. 2001; 98: 5728-33
• Display abstract

• The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes is how they interact with DNA, particularly in the large genomes of higher eukaryotes. Here, we report the characterization of BAP111, a BRM-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the BRM complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, we found that the majority of BAP111 protein in embryos is associated with the BRM complex. Furthermore, heterozygosity for BAP111 enhanced the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo.

• van Beest M et al.
• Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs.
• J Biol Chem. 2000; 275: 27266-73
• Display abstract

• Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment of promoter elements controlled by the yeast genes ste11 and Rox1 has indicated strict conservation of a larger DNA motif. By site selection, we identify a highly specific 12-base pair motif for Ste11, AGAACAAAGAAA. Similarly, we show that Tcf1, MatMc, and Sox4 bind unique, highly specific DNA motifs of 12, 12, and 10 base pairs, respectively. Footprinting with a deletion mutant of Ste11 reveals a novel interaction between the 3' base pairs of the extended DNA motif and amino acids C-terminal to the HMG domain. The sequence-specific interaction of Ste11 with these 3' base pairs contributes significantly to binding and bending of the DNA motif.

• Slama-Schwok A et al.
• Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.
• Biophys J. 2000; 78: 2543-59
• Display abstract

• Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation.

• Kamachi Y, Uchikawa M, Kondoh H
• Pairing SOX off: with partners in the regulation of embryonic development.
• Trends Genet. 2000; 16: 182-7
• Display abstract

• The SOX family of high-mobility group (HMG) domain proteins has recently been recognized as a key player in the regulation of embryonic development and in the determination of the cell fate. In the case of certain SOX proteins, they regulate the target genes by being paired off with specific partner factors. This partnering might allow SOX proteins to act in a cell-specific manner, which is key to their role in cell differentiation. The focus of this article is the mechanism of action of SOX proteins, in particular, how SOX proteins specifically pair off with respective partner factors and, as a consequence, select distinct sets of genes as their regulatory targets.

• Bianchi ME, Beltrame M
• Upwardly mobile proteins. Workshop: the role of HMG proteins in chromatin structure, gene expression and neoplasia.
• EMBO Rep. 2000; 1: 109-14

• Jamieson ER, Lippard SJ
• Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA.
• Biochemistry. 2000; 39: 8426-38
• Display abstract

• High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.

• Stros M, Muselikova E
• A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions.
• J Biol Chem. 2000; 275: 35699-707
• Display abstract

• HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.

• Dow LK, Jones DN, Wolfe SA, Verdine GL, Churchill ME
• Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.
• Biochemistry. 2000; 39: 9725-36
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• HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.

• Murphy FV 4th, Churchill ME
• Nonsequence-specific DNA recognition: a structural perspective.
• Structure Fold Des. 2000; 8: 839-839
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• Chromosomal proteins that form essential architectural components of chromatin bind and bend DNA with an intrinsic low degree of sequence preference. Comparisons made between two recently determined structures of high mobility group (HMG) protein-DNA complexes and other nonsequence-specific protein-DNA complexes reveal the structural basis of this important mode of DNA binding.

• Lee KB, Thomas JO
• The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal.
• J Mol Biol. 2000; 304: 135-49
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• The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous "HMG-box" DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.

• Xin H, Taudte S, Kallenbach NR, Limbach MP, Zitomer RS
• DNA binding by single HMG box model proteins.
• Nucleic Acids Res. 2000; 28: 4044-50
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• The HMG1/2 family is a large group of proteins that share a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg(2+). In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg(2+) is present in the nucleus and these proteins are expressed at a very high level.

• Benevides JM, Chan G, Lu XJ, Olson WK, Weiss MA, Thomas GJ Jr
• Protein-directed DNA structure. I. Raman spectroscopy of a high-mobility-group box with application to human sex reversal.
• Biochemistry. 2000; 39: 537-47
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• Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major-groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal.

• Rajski SR, Williams RM
• Observations on the covalent cross-linking of the binding domain (BD) of the high mobility group I/Y (HMG I/Y) proteins to DNA by FR66979.
• Bioorg Med Chem. 2000; 8: 1331-42
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• FR66979, a drug closely related to the mitomycin C class of antitumor antibiotics, is shown to covalently cross-link DNA to the DNA-binding domain of the High Mobility Group I/Y (HMG I/Y) DNA-binding proteins in the minor groove.

• Travers A
• Recognition of distorted DNA structures by HMG domains.
• Curr Opin Struct Biol. 2000; 10: 102-9
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• Recent biochemical and structural studies have shown that the preferential recognition of distorted DNA structures, including DNA bulges, four-way junctions and cis-platinated DNA, by HMG domains is dependent on residues immediately preceding the second alpha helix of the L-shaped HMG domain.

• Tang L, Li J, Katz DS, Feng JA
• Determining the DNA bending angle induced by non-specific high mobility group-1 (HMG-1) proteins: a novel method.
• Biochemistry. 2000; 39: 3052-60
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• To study the DNA bending induced by non-sequence-specific HMG-1 domain proteins, we have engineered a fusion protein linking the yeast NHP6A with a sequence-specific DNA binding domain, the DNA binding domain of the Hin recombinase, Hin-DBD. A series of biochemical experiments were carried out to characterize the DNA binding property of this fusion protein. Our data showed that the fusion protein not only specifically recognizes a DNA fragment containing the Hin-DBD binding site, but also binds DNA with a higher affinity in comparison with either domain alone. Both domains of the fusion protein are bound to the DNA in juxtaposition. Permutation assays showed that the fusion protein induced a DNA bending at the site of NHP6A binding by an estimated value of 63 degrees. We believe that this experimental design provides an effective vehicle to determine the DNA bending induced by nonspecific HMG-1 proteins.

• Bullejos M, Sanchez A, Burgos M, Jimenez R, Diaz De La Guardia R
• The SRY gene HMG-box in micro- and megabats.
• Cytogenet Cell Genet. 2000; 88: 30-4
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• Sex determination in mammals is controlled by the Y-linked SRY gene, which encodes a transcription factor with a DNA-binding motif of the HMG type. The only conserved region in this gene is the HMG-box, whose nucleotide sequence is currently available in a number of mammalian taxa. However, nothing is known about this gene in bats. Here, we report partial sequences of the SRY HMG-box from four microbat and four megabat species. We used the SRY HMG- box sequences from micro- and megabats to test the phylogenetic relationships between microbats, megabats, and primates. In maximum parsimony and maximum-likelihood trees, mega- and microbat branches start in the same internal node, which is consistent with a monophyletic origin of this mammalian group.

• Hill DA, Pedulla ML, Reeves R
• Directional binding of HMG-I(Y) on four-way junction DNA and the molecular basis for competitive binding with HMG-1 and histone H1.
• Nucleic Acids Res. 1999; 27: 2135-44
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• Histone H1, HMG-1 and HMG-I(Y) are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both HMG-1 and histone H1 on 4H DNA and compared these contacts with those previously described for HMG-I(Y) on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the HMG-I(Y) and HMG-1 proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of histone H1 bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized HMG-I(Y) protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of HMG-I(Y), histone H1 and HMG-1 in biological processes such as genetic recombination and retroviral integration.

• Wisniewski JR, Krohn NM, Heyduk E, Grasser KD, Heyduk T
• HMG1 proteins from evolutionary distant organisms distort B-DNA conformation in similar way.
• Biochim Biophys Acta. 1999; 1447: 25-34
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• The abundant high-mobility group proteins 1/2 (HMG1/2) represent a group of potent architectural elements of chromatin. They are able to induce strong bends and untwist DNA. Here, we compared the abilities of diverse HMG1 proteins to distort the B-DNA conformation of 30-base pair DNA fragment. The DNA bending was measured in solution by monitoring fluorescence resonance energy transfer between fluorescence probes attached to opposite ends of the DNA fragment. Various insect and plant proteins which differ in size, in composition of their HMG1-box domains (HMG1-BD), and in composition of the N- and the C-terminally flanking regions were analyzed in these experiments. Despite these structural differences the extent of the induced changes in DNA conformation upon binding to various proteins was similar, as the estimated bend angle was 150+/-20 degrees for all the tested proteins. Our results suggest that a set of highly conserved residues stabilizing the tertiary structure of the HMG1-BD mainly determines the extent of DNA bending in the complex. Even extended positively charged regions flanking the HMG1-BD are apparently not able to influence this conformational distortion of DNA.

• Ohndorf UM, Rould MA, He Q, Pabo CO, Lippard SJ
• Basis for recognition of cisplatin-modified DNA by high-mobility-group proteins.
• Nature. 1999; 399: 708-12
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• The anticancer activity of cis-diamminedichloroplatinum(II) (cisplatin) arises from its ability to damage DNA, with the major adducts formed being intrastrand d(GpG) and d(ApG) crosslinks. These crosslinks bend and unwind the duplex, and the altered structure attracts high-mobility-group domain (HMG) and other proteins. This binding of HMG-domain proteins to cisplatin-modified DNA has been postulated to mediate the antitumour properties of the drug. Many HMG-domain proteins recognize altered DNA structures such as four-way junctions and cisplatin-modified DNA, but until now the molecular basis for this recognition was unknown. Here we describe mutagenesis, hydroxyl-radical footprinting and X-ray studies that elucidate the structure of a 1:1 cisplatin-modified DNA/HMG-domain complex. Domain A of the structure-specific HMG-domain protein HMG1 binds to the widened minor groove of a 16-base-pair DNA duplex containing a site-specific cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] adduct. The DNA is strongly kinked at a hydrophobic notch created at the platinum-DNA crosslink and protein binding extends exclusively to the 3' side of the platinated strand. A phenylalanine residue at position 37 intercalates into a hydrophobic notch created at the platinum crosslinked d(GpG) site and binding of the domain is dramatically reduced in a mutant in which alanine is substituted for phenylalanine at this position.

• Banks GC, Mohr B, Reeves R
• The HMG-I(Y) A.T-hook peptide motif confers DNA-binding specificity to a structured chimeric protein.
• J Biol Chem. 1999; 274: 16536-44
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• Chromosomal translocations involving genes coding for members of the HMG-I(Y) family of "high mobility group" non-histone chromatin proteins (HMG-I, HMG-Y, and HMG-IC) have been observed in numerous types of human tumors. Many of these gene rearrangements result in the creation of chimeric proteins in which the DNA-binding domains of the HMG-I(Y) proteins, the so-called A.T-hook motifs, have been fused to heterologous peptide sequences. Although little is known about either the structure or biophysical properties of these naturally occurring fusion proteins, the suggestion has been made that such chimeras have probably assumed an altered in vivo DNA-binding specificity due to the presence of the A.T-hook motifs. To investigate this possibility, we performed in vitro "domain-swap" experiments using a model protein fusion system in which a single A. T-hook peptide was exchanged for a corresponding length peptide in the well characterized "B-box" DNA-binding domain of the HMG-1 non-histone chromatin protein. Here we report that chimeric A. T-hook/B-box hybrids exhibit in vitro DNA-binding characteristics resembling those of wild type HMG-I(Y) protein, rather than the HMG-1 protein. These results strongly suggest that the chimeric fusion proteins produced in human tumors as a result of HMG-I(Y) gene chromosomal translocations also retain A.T-hook-imparted DNA-binding properties in vivo.

• Lorenz M, Hillisch A, Payet D, Buttinelli M, Travers A, Diekmann S
• DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer.
• Biochemistry. 1999; 38: 12150-8
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• The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.

• Webb M, Thomas JO
• Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain.
• J Mol Biol. 1999; 294: 373-87
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• We have investigated the nature of the "structure-specific" binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-way junction DNA. AB didomain binding favours the open, planar form of the junction, as shown by reaction with potassium permanganate. Site-directed cleavage of the DNA by a 1, 10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain interacts with the widened minor groove at the centre, contacting one of the four strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolated B domain can, however, bind to the centre of the junction. The preferential binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably at least partly due to the higher positive surface potential in the DNA-binding region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged phosphates surrounding the central hole of the junction) and partly to differences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes.

• Wolffe AP
• Architectural regulations and Hmg1.
• Nat Genet. 1999; 22: 215-7

• Bustin M
• Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins.
• Mol Cell Biol. 1999; 19: 5237-46

• Churchill ME, Changela A, Dow LK, Krieg AJ
• Interactions of high mobility group box proteins with DNA and chromatin.
• Methods Enzymol. 1999; 304: 99-133

• Yoshioka Ki et al.
• Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein.
• Biochemistry. 1999; 38: 589-95
• Display abstract

• High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal. To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them. Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone. The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities. The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd

• Payet D, Hillisch A, Lowe N, Diekmann S, Travers A
• The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study.
• J Mol Biol. 1999; 294: 79-91
• Display abstract

• The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

• Jayaraman L, Moorthy NC, Murthy KG, Manley JL, Bustin M, Prives C
• High mobility group protein-1 (HMG-1) is a unique activator of p53.
• Genes Dev. 1998; 12: 462-72
• Display abstract

• The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. We report the identification of a factor from HeLa nuclear extracts that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. We demonstrate that recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro and also that HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53Delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. Finally, using transient transfection assays we show that HMG-1 can increase p53 and p53Delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures, and these data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA.

• Yamashita A et al.
• cDNA cloning of a novel rainbow trout SRY-type HMG box protein, rtSox23, and its functional analysis.
• Gene. 1998; 209: 193-200
• Display abstract

• We have isolated a cDNA clone for a new member of Sox genes, termed rtSox23, from a rainbow trout ovary cDNA library. rtSox23 mRNA was notably expressed in ovary and brain. rtSox23 contains a leucine zipper in addition to an SRY-type HMG box. Although the recombinant HMG box region protein of rtSox23 could bind to an AACAAT sequence, the full-length rtSox23 could form a homodimer and did not bind to the sequence. Furthermore, using a two-hybrid system, we have isolated a cDNA clone encoding a protein that bound to the leucine zipper region of rtSox23. This protein was the rainbow trout homologue of mouse nucleoporin p62, which is a component of the nuclear pore complex in nuclear envelope. The rainbow trout p62 mRNA was also prominent in ovary and brain. Taken together, these results suggest that the rainbow trout p62 associates with rtSox23 in vivo and modulates the function of rtSox23.

• Bewley CA, Gronenborn AM, Clore GM
• Minor groove-binding architectural proteins: structure, function, and DNA recognition.
• Annu Rev Biophys Biomol Struct. 1998; 27: 105-31
• Display abstract

• To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

• Stros M, Reich J
• Formation of large nucleoprotein complexes upon binding of the high-mobility-group (HMG) box B-domain of HMG1 protein to supercoiled DNA.
• Eur J Biochem. 1998; 251: 427-34
• Display abstract

• High-mobility group (HMG) 1 is a relatively highly abundant chromosomal protein with structural- rather than sequence-specific preference for binding to DNA. HMG1 has two highly related, folded domains A and B (HMG boxes), attached by a short basic region to an acidic C-terminal domain. We have studied binding of the B-domain of HMG1 protein and its mutants to supercoiled DNA by a gel-retardation assay and electron microscopy. Using a gel-retardation assay, we have demonstrated that HMG1 or HMG1 lacking the acidic C-terminal domain [i.e. HMG1(A+B) bi-domain], but not the isolated B-domain, could preferentially bind supercoiled over-relaxed closed circular or linear DNA. Mutational analysis of the HMG1 B-domain revealed that replacement of Lys96 of the extended N-terminal segment (and much less the neighboring Arg97) and Lys128 of helix II to glutamic acid severely impaired binding of the HMG box domain to supercoiled DNA. The latter mutation within helix II significantly decreased the alpha-helical content of the B-domain as revealed by circular dichroism. We have also shown that mutation of several residues within helix I of the B-domain, in particular Arg110, resulted in a diminished binding to supercoiled DNA as revealed by intensive smearing and reduced retardation of the protein/DNA complexes. These findings indicated that the extended N-terminus, helix I and helix II of the HMG1 B-domain are likely in contact with DNA. Electron microscopy revealed that the B-domain could bind to supercoiled DNA at higher HMG/DNA molar ratios as oligomeric protein beads with subsequent association of the beads into large nucleoprotein complexes from which many looped DNA molecules emerged. Most of the introduced mutations within all three helices of the B-domain (involving mainly basic and aromatic residues) abolished formation of the large nucleoprotein complexes even though the binding of the HMG box to supercoiled DNA was retained as revealed by a gel-retardation assay. A model for the interaction of the B-domain of HMG 1 with supercoiled DNA is presented and discussed.

• Kido S, Hiraoka Y, Ogawa M, Sakai Y, Yoshimura Y, Aiso S
• Cloning and characterization of mouse mSox13 cDNA.
• Gene. 1998; 208: 201-6
• Display abstract

• A novel SRY-related cDNA, mSox13, was isolated from a lambda phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence.

• Stros M
• DNA bending by the chromosomal protein HMG1 and its high mobility group box domains. Effect of flanking sequences.
• J Biol Chem. 1998; 273: 10355-61
• Display abstract

• HMG1 is an evolutionarily highly conserved chromosomal protein consisting of two folded DNA-binding domains, A and B ("high mobility group (HMG) boxes"), and an acidic C-terminal domain. Several lines of evidence suggest that previously reported sequence-independent DNA bending and looping by HMG1 and its HMG box domains might be important for the proposed role of the protein in transcription and recombination. We have used ligase-mediated circularization assays to investigate the contribution of the individual A and B HMG1 box domains and of the linker region between A/B- and B/C-domains, which flank the "minimal" B-domain (residues 92-162), to the ability of the HMG1 protein (residues 1-215) to bend DNA. Neither the minimal B-domain nor the minimal B-domain with a 7-residue N-terminal extension (85TKKKFKD91) bent the DNA. The attachment of an extra 18-residue C-terminal additional extension (residues 163-180) to the minimal B-domain had only a small effect on the ability of the HMG box to bend DNA. On the other hand, circularization assay with a B-domain having both 7-residue N-terminal and 18-residue C-terminal flanking sequences (residues 85-180) revealed a strong bending of the DNA, suggesting that both extensions are a prerequisite for efficient DNA bending by the B-domain. We have also shown that a single lysine residue (Lys90) in a short N-terminal sequence 90KD91 attached to the B-domain is sufficient for strong distortion of DNA by bending, provided that the B-domain is flanked by the 18-residue C-terminal flanking sequence. Although the DNA bending potential of HMG1 seems to be predominantly due to the B-domain flanked by basic sequences, covalent attachment of the A- and B-domains is necessary for efficient DNA flexure and the ability of the (A+B)-bidomain to bend DNA is further modulated in the native HMG1 protein by its acidic C-domain.

• Robbe K, Bonnefoy E
• Non-B-DNA structures on the interferon-beta promoter?
• Biochimie. 1998; 80: 665-71
• Display abstract

• The high mobility group (HMG) I protein intervenes as an essential factor during the virus induced expression of the interferon-beta (IFN-beta) gene. It is a non-histone chromatine associated protein that has the dual capacity of binding to a non-B-DNA structure such as cruciform-DNA as well as to AT rich B-DNA sequences. In this work we compare the binding affinity of HMGI for a synthetic cruciform-DNA to its binding affinity for the HMGI-binding-site present in the positive regulatory domain II (PRDII) of the IFN-beta promoter. Using gel retardation experiments, we show that HMGI protein binds with at least ten times more affinity to the synthetic cruciform-DNA structure than to the PRDII B-DNA sequence. DNA hairpin sequences are present in both the human and the murine PRDII-DNAs. We discuss in this work the presence of, yet putative, non-B-DNA structures in the IFN-beta promoter.

• Cremazy F, Soullier S, Berta P, Jay P
• Further complexity of the human SOX gene family revealed by the combined use of highly degenerate primers and nested PCR.
• FEBS Lett. 1998; 438: 311-4
• Display abstract

• SOX proteins contain a conserved HMG-related DNA-binding domain. They fulfill essential functions during the development of animals. Mutations in several SOX genes have been implicated in human diseases. We present here a new set of PCR primers designed to amplify a broad range of SOX HMG-box sequences. These primers facilitated the cloning of several new SOX HMG boxes from human genomic DNA, revealing unexpected complexity of the SOX gene family.

• P-ohler JR, Norman DG, Bramham J, Bianchi ME, Lilley DM
• HMG box proteins bind to four-way DNA junctions in their open conformation.
• EMBO J. 1998; 17: 817-26
• Display abstract

• The HMG box is an 80 amino acid domain found in a variety of eukaryotic chromosomal proteins and transcription factors. Binding to DNA is associated with recognition of structural distortion or manipulation of DNA structure. All the HMG box domains bind to four-way DNA junctions, which must therefore present some feature that is common to the binding targets of this wide variety of proteins. Since the four-way junction can itself adopt a variety of structures depending upon conditions, it is important to determine in which form it exists in complexes with HMG boxes. We find that a single HMG box domain is bound exclusively to the open square form of the junction and that conditions that stabilize the stacked X structure significantly lower affinity for the HMG box. We suggest that the HMG domain binds to one arm of the junction in the minor groove at the point of strand exchange and we present a model for the structure of the complex.

• Bianchi ME, Beltrame M
• Flexing DNA: HMG-box proteins and their partners.
• Am J Hum Genet. 1998; 63: 1573-7

• Dow LK, Changela A, Hefner HE, Churchill ME
• Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D.
• FEBS Lett. 1997; 414: 514-20
• Display abstract

• HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. During overexpression and purification of HMG-D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.

• Weiss MA, Ukiyama E, King CY
• The SRY cantilever motif discriminates between sequence- and structure-specific DNA recognition: alanine mutagenesis of an HMG box.
• J Biomol Struct Dyn. 1997; 15: 177-84
• Display abstract

• The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.

• Stemmer C, Ritt C, Igloi GL, Grimm R, Grasser KD
• Variability in Arabidopsis thaliana chromosomal high-mobility-group-1-like proteins.
• Eur J Biochem. 1997; 250: 646-52
• Display abstract

• The vertebrate high-mobility-group (HMG) protein HMG1 is an abundant non-histone protein which is considered as an architectural element in chromatin. In the monocotyledonous plant maize, four different HMG1-like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type. We have examined here the HMG1-like proteins of the dicotyledonous plant Arabidopsis thaliana. The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1-like proteins (now termed HMG alpha, HMG beta1/2, HMG gamma, HMG delta) from Arabidopsis indicates that the expression of multiple HMG1-like proteins is a general feature of (higher) plants. The Arabidopsis HMG1-like proteins contain an HMG domain as a common feature, but outside this conserved DNA-binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes. The five HMG1-like proteins were expressed in Escherichia coli and purified. They bind with somewhat different affinity to linear double-stranded DNA. The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA. Reverse-transcribed PCR suggested that the five HMG1-like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.

• Wisniewski JR, Hessler K, Claus P, Zechel K
• Structural and functional consequences of mutations within the hydrophobic cores of the HMG1-box domain of the Chironomus high-mobility-group protein 1a.
• Eur J Biochem. 1997; 243: 151-9
• Display abstract

• The high-mobility-group protein 1 box domain (HMG1-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three alpha-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trp14 of the Chironomus HMG1a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletion of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMG1a, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wild-type cHMG1a and [W14A]cHMG1a showed that the proteins unfold at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-8 M of the denaturant. Multiple contacts between the HMG1-BD and the DNA are probably responsible for the unusual stability of the complexes.

• Pohler JR, Lilley DM
• The interaction of HMG-box proteins with the four-way DNA junction.
• Biochem Soc Trans. 1997; 25: 647-647

• Heyduk E, Heyduk T, Claus P, Wisniewski JR
• Conformational changes of DNA induced by binding of Chironomus high mobility group protein 1a (cHMG1a). Regions flanking an HMG1 box domain do not influence the bend angle of the DNA.
• J Biol Chem. 1997; 272: 19763-70
• Display abstract

• High mobility group (HMG) proteins are thought to facilitate assembly of higher order chromatin structure through modulation of DNA conformation. In this work we investigate the bending of a 30-base pair DNA fragment induced by Chironomus HMG1 (cHMG1a), and HMGI (cHMGI) proteins. The DNA bending was measured in solution by monitoring the end-to-end distance between fluorescence probes attached to opposite ends of the DNA fragment. The distance was measured by fluorescence energy transfer using a novel europium chelate as a fluorescence donor. These measurements revealed that the end-to-end distance in the 30-base pair DNA was decreased from approximately 100 A in free DNA to approximately 50.5 A in cHMG1a. DNA complex. The most probable DNA bending angle consistent with these distance measurements is about 150 degrees. The deletion of the charged regulatory domains located close to the C terminus of the HMG1 box domain of cHMG1a protein had no effect on the induced bend angle. The ability to induce a large DNA bend distinguishes the cHMG1 from the cHMGI protein. Only small perturbation of the DNA conformation was observed upon binding of the cHMGI protein. A strong DNA bending activity of cHMG1a and its relative abundance in the cell suggests that this protein plays a very important role in modulation of chromatin structure.

• Payet D, Travers A
• The acidic tail of the high mobility group protein HMG-D modulates the structural selectivity of DNA binding.
• J Mol Biol. 1997; 266: 66-75
• Display abstract

• HMG-D is one of the Drosophila counterparts of the vertebrate HMG1/2 class of abundant chromosomal proteins and contains three domains: an HMG domain followed by a basic region and a short acidic carboxyterminal tail. We show that the HMG domain of HMG-D does not bind to deformed DNA structures such as DNA bulges, cis-platinated DNA or four-way junctions but does bind tightly to DNA microcircles, suggesting that in vivo the natural ligands of this domain are tightly bent DNA loops. The flanking basic region substantially increases the DNA-binding activity of the HMG domain to DNA ligands other than microcircles. We demonstrate that the acidic tail alters the structural selectivity of DNA binding by increasing the affinity for deformed DNA and decreasing the affinity for linear B-DNA. Finally, we show that the acidic tail increases the efficiency of constraining preformed negative supercoils but conversely decreases the efficiency of supercoiling relaxed DNA in the presence of topoisomerase I.

• Grasser KD, Grimm R, Ritt C
• Maize chromosomal HMGc. Two closely related structure-specific DNA-binding proteins specify a second type of plant high mobility group box protein.
• J Biol Chem. 1996; 271: 32900-6
• Display abstract

• The chromosomal high mobility group (HMG) proteins are small and abundant non-histone proteins common to eukaryotes. We have purified the maize HMGc protein from immature kernels and characterized it by mass spectrometry and amino acid sequence analysis. HMGc could be resolved into two similar proteins by reversed phase chromatography. Cloning and characterization of the corresponding cDNAs revealed that they encode two closely related maize HMGc proteins, now termed HMGc1 and HMGc2. Their theoretical masses of 15,316 and 15,007 Da are >300 Da lower than the masses determined for the proteins purified from maize, indicating post-translational modifications of the proteins. Despite sequence similarity to maize HMGa (and previously described homologous proteins of other species) amino acid sequence alignments reveal that HMGc is in several conserved regions distinct from these proteins. Consequently, we have identified a novel type of plant protein containing an HMG box DNA binding domain and belonging to the HMG1 protein family. HMGc1 and HMGc2 were expressed in Escherichia coli, purified to homogeneity, and analyzed for their DNA binding properties. They proved to bind to DNA structure-specifically since they formed complexes with DNA minicircles at concentrations approximately 100-fold lower than the concentrations required to form complexes with linear fragments of identical sequence. Furthermore, HMGc1 and HMGc2 can constrain negative superhelical turns in plasmid DNA.

• Yoshida M
• [HMG box proteins: general architectural elements in the assembly of active transcription complex]
• Seikagaku. 1996; 68: 1829-34

• Zappavigna V, Falciola L, Helmer-Citterich M, Mavilio F, Bianchi ME
• HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation.
• EMBO J. 1996; 15: 4981-91
• Display abstract

• High mobility group protein 1 (HMG1) is a non-histone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model. HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain. HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.

• Suda T, Mishima Y, Takayanagi K, Asakura H, Odani S, Kominami R
• A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.
• Nucleic Acids Res. 1996; 24: 4733-40
• Display abstract

• The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.

• Lnenicek-Allen M, Read CM, Crane-Robinson C
• The DNA bend angle and binding affinity of an HMG box increased by the presence of short terminal arms.
• Nucleic Acids Res. 1996; 24: 1047-51
• Display abstract

• The HMG box of human LEF-1 (hLEF-1, formerly TCF1alpha) has been expressed in four forms: a parent box of 81 amino acids and constructs having either a 10 amino acid C-terminal extension, a 9 amino acid N-terminal extension, or both. These four species have been compared for DNA binding and bending ability using a 28 bp recognition sequence from the TCR alpha-chain enhancer. In the bending assay, whereas the parent box and that with the N-terminal extension bent the DNA by 57/58 degrees, the box extended at the C-terminus bent the DNA by 77/78 degrees, irrespective of the presence or absence of the N-terminal extension. A 6- fold increase in DNA affinity also resulted from addition of both terminal extensions. These observations redefine the functional boundaries of the HMG box. The structure of a mouse LEF-1/DNA complex recently published [Love et al. (1995) Nature 376, 791-795] implies that the higher DNA affinity and in particular the increased bend angle observed are consequences, at least in part, of the C-terminal extension spanning the major groove on the inside of the DNA bend.

• Travers AA
• Reading the minor groove.
• Nat Struct Biol. 1995; 2: 615-8

• Baxevanis AD, Landsman D
• The HMG-1 box protein family: classification and functional relationships.
• Nucleic Acids Res. 1995; 23: 1604-13
• Display abstract

• The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts.

• Teo SH, Grasser KD, Thomas JO
• Differences in the DNA-binding properties of the HMG-box domains of HMG1 and the sex-determining factor SRY.
• Eur J Biochem. 1995; 230: 943-50
• Display abstract

• High-mobility-group protein 1 (HMG1) is an abundant, non-sequence-specific, chromosomal protein with two homologous, HMG-box, DNA-binding domains, A and B, and an acidic tail. The HMG-box motif also occurs, as a single copy, in some sequence-specific transcription factors, e.g. the sex-determining factor, SRY. We have investigated whether or not there are differences in the DNA-binding properties of the isolated A and B HMG-box domains of HMG1 and SRY and whether, in the case of A and B, there might also be differences due to different sequence contexts within the native protein. The basic regions that flank the HMG1 B box, giving B', enhance its DNA-binding, supercoiling and DNA-bending activities, and promote the self-association of the DNA-bound B-box. All the HMG-box domains bind with structure specificity to four-way junctions, but the structure selectivity is significantly greater for A and the SRY box than for the HMG1 B or B' domains, as judged by competition with excess plasmid DNA. The domains self-associate to different extents on supercoiled DNA and this may explain differences in the ability to discriminate between four-way junctions and supercoiled DNA. The HMG1 A, B and B' domains constrain negative superhelical turns in DNA, but the SRY HMG box does not. Only the full B domain (B') bends DNA in a ligase-mediated circularisation assay; the minimal B box, the A domain and the SRY box do not. Thus, despite a common global fold, the HMG box appears to have been adapted to various functions in different protein contexts.

• Teo SH, Grasser KD, Hardman CH, Broadhurst RW, Laue ED, Thomas JO
• Two mutations in the HMG-box with very different structural consequences provide insights into the nature of binding to four-way junction DNA.
• EMBO J. 1995; 14: 3844-53
• Display abstract

• Mutation of the highly conserved tryptophan residue in the A-domain HMG-box of HMG1 largely, but not completely, destroys the protein tertiary structure and abolishes its supercoiling ability, but does not abolish structure-specific DNA binding to four-way junctions. Circular dichroism shows that the protein has some residual alpha-helix (< 10%) and does not re-fold in the presence of DNA. Structure-specific DNA binding might therefore be a property of some primary structure element, for example the N-terminal extended strand, which even in the unfolded protein would be held in a restricted conformation by two, largely trans, X-Pro peptide bonds. However, mutation of P5 or P8 of the A-domain to alanine does not abolish the formation of the (first) complex in a gel retardation assay, which probably arises from binding to the junction cross-over, although the P8 mutation does affect the formation of higher complexes which may arise from binding to the junction arms. Since mutation of P8 in the W49R mutant has no effect on structure-specific junction binding, we propose that some residual alpha-helix in the protein might be involved, implicating this element in the interactions of HMG-boxes generally with DNA.

• Vriz S, Lovell-Badge R
• The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain.
• Gene. 1995; 153: 275-6
• Display abstract

• A cDNA encoding a zebrafish Sox protein (Sry-type high-mobility-group box) was isolated and sequenced. The sequence within the HMG box is close to those of the B subfamily comprising mouse Sox-1, Sox-2, Sox-3 and Sox-14. While much of the rest of the zebrafish protein is unique, there are blocks of amino acids showing considerable identity with regions of Sox-1, -2 and -3. These domains may represent conserved parts of the protein, required for interaction with other proteins, and strengthen the assignment of the zebrafish gene, termed Zf-sox 19, to the B subfamily.

• Wagner JP, Quill DM, Pettijohn DE
• Increased DNA-bending activity and higher affinity DNA binding of high mobility group protein HMG-1 prepared without acids.
• J Biol Chem. 1995; 270: 7394-8
• Display abstract

• Recently, DNA ring closure assays showed that high mobility group protein HMG-1 and its close homolog HMG-2 mediate sequence-independent DNA flexion. This DNA-bending activity appears to be central to at least some of the recently elucidated functions of HMG-1/2, such as the enhancement of progesterone receptor DNA binding. Here we show that standard purification procedures utilizing perchloric and trichloroacetic acid can produce HMG-1 significantly deficient in its abilities to bind and bend double-stranded DNA, while acid-independent methods purify HMG-1 that is superior in these respects. Significant losses of DNA ring closure activity were seen upon limited 2-5-h exposures of nonacid-purified HMG-1/2 to perchloric acid and/or trichloroacetic acid. Measurements of the apparent DNA dissociation binding constant (Kd(app)) of acid-extracted preparations of HMG-1 gave a wide range of values, and only those preparations demonstrating little DNA ring closure activity had Kd values near the previously published value (approximately 10(-6) M). The highest ring closure activities and lowest Kd(app) (< 3 x 10(-9) M) were obtained for HMG-1 purified without acids. These combined results support the use of alternative, non-acid purification procedures for preserving the DNA-bending activity of HMG-1/2 and suggest that past procedures utilizing acids have led to an underestimation of the affinity of HMG-1 for DNA.

• Werner MH, Huth JR, Gronenborn AM, Clore GM
• Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex.
• Cell. 1995; 81: 705-14
• Display abstract

• The solution structure of the specific complex between the high mobility group (HMG) domain of SRY (hSRY-HMG), the protein encoded by the human testis-determining gene, and its DNA target site in the promoter of the mullerian inhibitory substance gene has been determined by multidimensional NMR spectroscopy. hSRY-HMG has a twisted L shape that presents a concave surface (made up of three helices and the N- and C-terminal strands) to the DNA for sequence-specific recognition. Binding of hSRY-HMG to its specific target site occurs exclusively in the minor groove and induces a large conformational change in the DNA. The DNA in the complex has an overall 70 degrees-80 degrees bend and is helically unwound relative to classical A- and B-DNA. The structure of the complex reveals the origin of sequence-specific binding within the HMG-1/HMG-2 family and provides a framework for understanding the effects of point mutations that cause 46X,Y sex reversal at the atomic level.

• Locker D, Decoville M, Maurizot JC, Bianchi ME, Leng M
• Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.
• J Mol Biol. 1995; 246: 243-7
• Display abstract

• The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.

• Love JJ, Li X, Case DA, Giese K, Grosschedl R, Wright PE
• Structural basis for DNA bending by the architectural transcription factor LEF-1.
• Nature. 1995; 376: 791-5
• Display abstract

• Lymphoid enhancer-binding factor (LEF-1) and the closely related T-cell factor 1 (TCF-1) are sequence-specific and cell-type-specific DNA-binding proteins that play important regulatory roles in organogenesis and thymocyte differentiation. LEF-1 participates in regulation of the enhancer associated with the T cell receptor (TCR)-alpha gene by inducing a sharp bend in the DNA and facilitating interactions between Ets-1, PEBP2-alpha, and ATF/CREB, transcription factors bound at sites flanking the LEF-1 site. It seems that LEF-1 plays an architectural role in the assembly and function of this regulatory nucleoprotein complex. LEF-1 recognizes a specific nucleotide sequence through a high-mobility-group (HMG) domain. Proteins containing HMG domains bind DNA in the minor groove, bend the double helix, and recognize four-way junctions and other irregular DNA structures. Here we report the solution structure of a complex of the LEF-1 HMG domain and adjacent basic region with its cognate DNA. The structure reveals the HMG domain bound in the widened minor groove of a markedly distorted and bent double helix. The basic region binds across the narrowed major groove and contributes to DNA recognition.

• Churchill ME, Jones DN, Glaser T, Hefner H, Searles MA, Travers AA
• HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.
• EMBO J. 1995; 14: 1264-75
• Display abstract

• The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.

• Siino JS, Nissen MS, Reeves R
• Replacement of conserved threonines by alanine residues in high mobility group protein HMG-I(Y): effect on DNA binding affinity.
• Biochem Biophys Res Commun. 1995; 207: 497-507
• Display abstract

• A threonine residue at the beginning of each DNA-binding domain of HMG-I (residue numbers 21, 53, and 78) is conserved among mammalian species and proposed to help stabilize the A.T-hook DNA-binding motif. Phosphorylation of threonines number 53 and 78 of human HMG-I(Y) both in vivo and in vitro leads to a 20 fold reduction in the proteins DNA binding affinity. Recombinant human HMG-I proteins were engineered to contain alanine instead of the conserved threonine in each DNA-binding domain. The DNA dissociation constant of each protein was assayed at various salt concentrations by competition with the fluorescent dye Hoechst 33258 for an AT-rich DNA substrate. Replacement of these threonines did not affect the equilibrium binding of these proteins to DNA as compared with wild-type HMG-I and HMG-Y. Molecular modelling of analogous peptides supported this finding. We conclude that these threonines are not directly important for A.T-hook DNA-binding and are conserved phosphorylation sites for down regulation of DNA binding by the A.T-hook motif in the HMG-I(Y) proteins.

• Peters R, King CY, Ukiyama E, Falsafi S, Donahoe PK, Weiss MA
• An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction.
• Biochemistry. 1995; 34: 4569-76
• Display abstract

• SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

• Bianchi ME, Lilley DM
• DNA--protein interactions. Applying a genetic cantilever.
• Nature. 1995; 375: 532-532

• Evans JN, Zajicek J, Nissen MS, Munske G, Smith V, Reeves R
• 1H and 13C NMR assignments and molecular modelling of a minor groove DNA-binding peptide from the HMG-I protein.
• Int J Pept Protein Res. 1995; 45: 554-60
• Display abstract

• The HMG-I subfamily of high mobility group (HMG) chromatin proteins consists of DNA-binding proteins that preferentially bind to stretches of A.T-rich sequence both in vitro and in vivo. Recently, members of the HMG-I family have been suggested to bind in vitro to the narrow minor groove of A.T-DNA by means of an 11 amino acid peptide binding domain (BD) which, because of its predicted structure, is called the 'A.T-hook motif' [Reeves, R. & Nissen, M. (1990) J. Biol. Chem. 265, 8573-8582], and would appear to be crescent-shaped. A BD peptide with 13 amino-acid residues was synthesized and examined by proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. The peptide contains four proline residues, and on the basis of NOEs and 13C chemical shifts was found to exist in an all-trans conformation. Molecular modelling based on this result provides evidence for a dynamic equilibrium between turn-like conformations in solution, the most populated of which is likely to be an S-shaped conformer, on the basis of amide exchange data.

• Chow CS, Barnes CM, Lippard SJ
• A single HMG domain in high-mobility group 1 protein binds to DNAs as small as 20 base pairs containing the major cisplatin adduct.
• Biochemistry. 1995; 34: 2956-64
• Display abstract

• Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.

• Hardman CH, Broadhurst RW, Raine AR, Grasser KD, Thomas JO, Laue ED
• Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy.
• Biochemistry. 1995; 34: 16596-607
• Display abstract

• HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain. Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns. They show qualitatively similar properties with quantitative differences. We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold [Weir et al. (1993) EMBO J. 12, 1311-1319]. We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy. The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D [Jones et al. (1994) Structure 2, 609-627]. There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains [Teo et al. (1995) Eur. J. Biochem. 230, 943-950] and will be the subject of further investigation. NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II. These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes [Love et al. (1995) Nature 376, 791-795; Werner et al. (1995) Cell 81, 705-714].

• van Houte LP, Chuprina VP, van der Wetering M, Boelens R, Kaptein R, Clevers H
• Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4.
• J Biol Chem. 1995; 270: 30516-24
• Display abstract

• Two groups of HMG box proteins are distinguished. Proteins in the first group contain multiple HMG boxes, are non-sequence-specific, and recognize structural features as found in cruciform DNA and cross-over DNA. The abundant chromosomal protein HMG-1 belongs to this subgroup. Proteins in the second group carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. A solution structure for the non-sequence-specific C-terminal HMG box of HMG-1 has recently been proposed. Now, we report the solution structure of the sequence-specific HMG-box of the SRY-related protein Sox-4. NMR analysis demonstrated the presence of three alpha-helices (Val10-Gln22, Glu30-Leu41 and Phe50-Tyr65) connected by loop regions (Ser23-Ala49 and Leu42-Pro49). Helices I and II are positioned in an antiparallel mode and form one arm of the HMG box. Helix III is less rigid, makes an average angle of about 90 degrees with helices I and II, and constitutes the other arm of the molecule. As in HMG1B, the overall structure of the Sox-4 HMG box is L-shaped and is maintained by a cluster of conserved, mainly aromatic residues.

• Grasser KD, Krech AB, Feix G
• The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility.
• Plant J. 1994; 6: 351-8
• Display abstract

• The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.

• Wisniewski JR, Ghidelli S, Steuernagel A
• Region of insect high mobility group (HMG) 1 protein homologous to helix 2 of the rat HMG1-b box is in close contact with DNA.
• J Biol Chem. 1994; 269: 29261-4
• Display abstract

• The dipteran insects Chironomus and Drosophila have high mobility group (HMG) 1 proteins that are similar to mammalian HMG1 but contain one instead of two HMG1 boxes. The interaction of the Chironomus HMG1 proteins cHMG1a and cHMG1b with double-stranded and four-way junction DNA was analyzed by investigating the accessibility of defined sequences of the HMG1 box to specific antibodies within the DNA-protein complex in vitro. Antibody epitopes located in the three helices and in the turn between helices 1 and 2 of the HMG1 box were identified on a membrane onto which 90 decapeptides with overlapping sequences spanning the entire sequence of cHMG1a had been bound. Monospecific antibodies directed against selected epitopes were purified from a polyclonal antiserum by affinity chromatography. Helices 1 and 3 as well as the turn between helices 1 and 2 were found to be accessible to antibodies in the complex. In contrast, antibodies recognizing an epitope on putative helix 2 of cHMG1a and cHMG1b were unable to produce supershifts on gels of the DNA-protein complexes with both DNAs. These data suggest that helix 2 of the HMG1 box of proteins cHMG1a and cHMG1b is essentially responsible for contacts with DNA.

• Gaillard C, Strauss F
• Association of poly(CA).poly(TG) DNA fragments into four-stranded complexes bound by HMG1 and 2.
• Science. 1994; 264: 433-6
• Display abstract

• The tandemly repeated DNA sequence poly(CA).poly(TG) is found in tracts up to 60 base pairs long, dispersed at thousands of sites throughout the genomes of eukaryotes. Double-stranded DNA fragments containing such sequences associated spontaneously with each other in vitro, in the absence of protein, forming stable four-stranded structures that were detected by gel electrophoresis and electron microscopy. These structures were recognized specifically by the nuclear nonhistone high mobility group (HMG) proteins 1 and 2 as evidenced by gel retardation. Such sequence-specific complexes might be involved in vivo in recombination or other processes requiring specific association of two double-stranded DNA molecules.

• Read CM, Cary PD, Preston NS, Lnenicek-Allen M, Crane-Robinson C
• The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.
• EMBO J. 1994; 13: 5639-46
• Display abstract

• To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.

• Stros M, Reich J, Kolibalova A
• Calcium binding to HMG1 protein induces DNA looping by the HMG-box domains.
• FEBS Lett. 1994; 344: 201-6
• Display abstract

• Electron microscopy has shown that non-histone chromosomal HMG1 could induce DNA looping or compaction in the presence (but not in the absence) of Ca2+. The effect of calcium on DNA looping and compaction was interpreted as calcium binding to the acidic C-domain of HMG1. Both individual DNA-binding HMG1-box domains A and B were found to be involved in DNA looping and compaction. Treatment of HMG1 with a thiol-specific reagent, N-ethylmaleimide, inhibited the ability of the protein to induce DNA looping and compaction but not the electrostatic interaction with DNA. These results indicated that cysteine-sulfhydryl groups of the HMG1-box domains A and B are specifically involved in DNA looping and compaction, and that in the absence of calcium the acidic C-domain down-regulates these effects by modulation of the DNA-binding properties of the HMG1-box domains.

• Geierstanger BH, Volkman BF, Kremer W, Wemmer DE
• Short peptide fragments derived from HMG-I/Y proteins bind specifically to the minor groove of DNA.
• Biochemistry. 1994; 33: 5347-55
• Display abstract

• Short peptides derived from chromosomal proteins have previously been proposed to bind specifically to the minor groove of A,T-rich DNA [for a review, see M. E. A. Churchill and A. A. Travers (1991) Trends Biochem. Sci. 16, 92-97]. Using NMR spectroscopy, we investigated the DNA binding of SPRKSPRK, which is one such A,T-specific motif. Under the conditions studied SPRKSPRK interacts only nonspecifically with d(CGCAAAAAAGGC).d(GCCTTTTTTGCG). The peptides TPKRPRGRPKK, PRGRPKK, and PRGRP derived from the non-histone chromosomal protein HMG-I/Y, however, bind specifically to the central A,T sites of d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2. 2D NOE measurements show that the RGR segment of each peptide is in contact with the minor groove. The arginine side chains and the peptide backbone are buried deep in the minor groove, in a fashion generally similar to the antibiotic netropsin. Under the same conditions the peptide PKGKP does not interact with the same oligonucleotide duplexes, indicating that the arginine guanidinium groups are major determinants of the A,T specificity.

• Falciola L, Murchie AI, Lilley DM, Bianchi M
• Mutational analysis of the DNA binding domain A of chromosomal protein HMG1.
• Nucleic Acids Res. 1994; 22: 285-92
• Display abstract

• We have mutated several residues of the first of the two HMG-boxes of mammalian HMG1. Some mutants cannot be produced in Escherichia coli, suggesting that the peptide fold is grossly disrupted. A few others can be produced efficiently and have normal DNA binding affinity and specificity; however, they are more sensitive towards heating and chaotropic agents than the wild type polypeptide. Significantly, the mutation of the single most conserved residue in the rather diverged HMG-box family falls in this 'in vitro temperature-sensitive' category, rather than in the non-folded category. Finally, two other mutants have reduced DNA binding affinity but unchanged binding specificity. Overall, it appears that whenever the HMG-box can fold, it will interact specifically with kinked DNA.

• Stros M, Stokrova J, Thomas JO
• DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.
• Nucleic Acids Res. 1994; 22: 1044-51
• Display abstract

• We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.

• Postnikov YV, Lehn DA, Robinson RC, Friedman FK, Shiloach J, Bustin M
• The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.
• Nucleic Acids Res. 1994; 22: 4520-6
• Display abstract

• Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

• Bianchi ME
• Prokaryotic HU and eukaryotic HMG1: a kinked relationship.
• Mol Microbiol. 1994; 14: 1-5
• Display abstract

• HU and IHF proteins have long been considered the prokaryotic analogues of eukaryotic histones. Their ability to bend DNA, however, is distinctly similar to that of eukaryotic HMG-box proteins, a recently identified family of chromatin components and transcription factors. In some conditions, HU and HMG1-like proteins can even be swapped, both in vitro and in vivo. In spite of this, HU/IHF and HMG-box proteins are not evolutionarily related, and represent two independent solutions for the same biochemical problem.

• Wolffe AP
• Architectural transcription factors.
• Science. 1994; 264: 1100-1

• Jones DN et al.
• The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.
• Structure. 1994; 2: 609-27
• Display abstract

• BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

• Ner SS, Travers AA, Churchill ME
• Harnessing the writhe: a role for DNA chaperones in nucleoprotein-complex formation.
• Trends Biochem Sci. 1994; 19: 185-7

• Giese K, Pagel J, Grosschedl R
• Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.
• Proc Natl Acad Sci U S A. 1994; 91: 3368-72
• Display abstract

• The mammalian sex-determining gene SRY (sex-determining region on Y chromosome) encodes a member of the high mobility group (HMG) family of regulatory proteins. The HMG domain of the SRY protein represents a DNA binding motif that displays rather unusually weak evolutionary conservation of amino acids between human and mouse sequences. Together with the previous finding that the human (h) SRY gene is unable to induce a male phenotype in genetically female transgenic mice, these observations raise questions concerning the DNA binding properties of SRY proteins. Here, we present data that indicate that the DNA binding and bending properties of the HMG domains of murine (m) SRY and hSRY differ from each other. In comparison, mSRY shows more-extensive major-groove contacts with DNA and a higher specificity of sequence recognition than hSRY. Moreover, the extent of protein-induced DNA bending differs from the HMG domains of hSRY and mSRY. These differences in DNA binding by hSRY and mSRY may, in part, account for the functional differences observed with these gene products.

• Connor F et al.
• DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.
• Nucleic Acids Res. 1994; 22: 3339-46
• Display abstract

• Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.

• Chow CS, Whitehead JP, Lippard SJ
• HMG domain proteins induce sharp bends in cisplatin-modified DNA.
• Biochemistry. 1994; 33: 15124-30
• Display abstract

• Circularly permuted linear DNAs of approximately 100 bp were constructed containing the major adduct of the anticancer drug cisplatin, a cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link, at a specific site. Gel electrophoresis mobility shift assays with these probes were used to investigate the effects of binding of HMG domain proteins to the platinated DNAs. The site-specifically platinated duplexes were recognized by six different HMG domain proteins--HMG1, mtTFA, Ixr1, and HMG domains from HMG1 (domain B), mSRY, and LEF-1--with comparable binding affinities (Kd approximately 10(-6) to 10(-7) M). In the presence of the HMG domain proteins, the platinated DNAs were bent significantly more than in their absence, the values being 86 +/- 2 degrees, 87-90 +/- 5 degrees, and 68 +/- 6 degrees, respectively, for the proteins and 65-74 +/- 4 degrees, approximately 50 degrees, and 72 +/- 6 degrees, respectively, for the domains. The variability in bend angles suggests that, although the HMG domain proteins share a common ability to bend platinated DNA, specific contacts between the proteins and the platinated duplex are different. The assay further revealed the bend loci to be centered quite near the platinum adduct. The methodology employed in the present study should be generally applicable for synthesizing other small, circularly permuted, covalently modified DNAs which cannot otherwise be readily obtained.

• Wisniewski JR, Schulze E
• High affinity interaction of dipteran high mobility group (HMG) proteins 1 with DNA is modulated by COOH-terminal regions flanking the HMG box domain.
• J Biol Chem. 1994; 269: 10713-9
• Display abstract

• The cells of the dipteran insects Chironomus and Drosophila contain high mobility group (HMG) 1 proteins that are homologous to the HMG1 protein of mammals but comprise one instead of two DNA-binding HMG boxes. Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double strand and four-way junction DNA in a similar way at apparent dissociation constants in the range of 7.5-20 x 10(-9) M. Both proteins are monomeric and highly asymmetric molecules in solution. cHMG1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, and both show a frictional ratio of 1.5. Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is approximately 1.5 of the size found for the cHMG1b. Enzymatically and chemically prepared peptides of cHMG1a as well as bacterially expressed cHMG1a with terminal deletions and point substitutions showed that sequences flanking the folded domain that constitutes the HMG box are essential for the interaction of the HMG box with DNA. In particular, changes in the number of positive and negative charges, respectively, within basic and acidic domains modulated the DNA binding affinity of the cHMG1a protein. The alteration of fluorescence of the Trp residues suggest that this modulation is due to interaction of the acidic domain with the positively charged HMG box.

• Landsman D, Bustin M
• A signature for the HMG-1 box DNA-binding proteins.
• Bioessays. 1993; 15: 539-46
• Display abstract

• A diverse group of DNA-binding regulatory proteins share a common structural domain which is homologous to the sequence of a highly conserved and abundant chromosomal protein, HMG-1. Proteins containing this HMG-1 box regulate various cellular functions involving DNA binding, suggesting that the target DNA sequences share a common structural element. Members of this protein family exhibit a dual DNA-binding specificity: each recognizes a unique sequence as well as a common DNA conformation. The highly conserved HMG-1/-2 proteins may modulate the binding of other HMG-1 box proteins to bent DNA. We examine the structural and functional relationships between the proteins, identify their signature and describe common features of their target DNA elements.

• Griess EA, Rensing SA, Grasser KD, Maier UG, Feix G
• Phylogenetic relationships of HMG box DNA-binding domains.
• J Mol Evol. 1993; 37: 204-10
• Display abstract

• HMG boxes were initially identified as DNA-binding domains of the human RNA polymerase I (pol I) transcription factor hUBF and the animal high-mobility-group (HMG) protein family HMG1. Since then, numerous sequences of HMG-box-containing HMG proteins and other DNA-binding proteins from several species have become available. By sequence comparisons of a selected range of HMG boxes from these proteins and the construction of phylogenetic trees we show that the HMG box is highly conserved between DNA-binding proteins of organisms from all three eukaryotic kingdoms and that HMG boxes are linked by distinct evolutionary relationships. In addition, most HMG boxes display comparable hydropathy profiles and amino acid arrangements, which could serve as nuclear targeting sequences.

• Pil PM, Chow CS, Lippard SJ
• High-mobility-group 1 protein mediates DNA bending as determined by ring closures.
• Proc Natl Acad Sci U S A. 1993; 90: 9465-9
• Display abstract

• High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.

• Laudet V, Stehelin D, Clevers H
• Ancestry and diversity of the HMG box superfamily.
• Nucleic Acids Res. 1993; 21: 2493-501
• Display abstract

• The HMG box is a novel type of DNA-binding domain found in a diverse group of proteins. The HMG box superfamily comprises a.o. the High Mobility Group proteins HMG1 and HMG2, the nucleolar transcription factor UBF, the lymphoid transcription factors TCF-1 and LEF-1, the fungal mating-type genes mat-Mc and MATA1, and the mammalian sex-determining gene SRY. The superfamily dates back to at least 1,000 million years ago, as its members appear in animals, plants and yeast. Alignment of all known HMG boxes defined an unusually loose consensus sequence. We constructed phylogenetic trees connecting the members of the HMG box superfamily in order to understand their evolution. This analysis led us to distinguish two subfamilies: one comprising proteins with a single sequence-specific HMG box, the other encompassing relatively non sequence-specific DNA-binding proteins with multiple HMG boxes. By studying the extent of diversification of the superfamily, we found that the speed of evolution was very different within the various groups of HMG-box containing factors. Comparison of the evolution of the two boxes of ABF2 and of mtTF1 implied different diversification models for these two proteins. Finally, we provide a tree for the highly complex group of SRY-like ('Sox' genes), clustering at least 40 different loci that rapidly diverged in various animal lineages.

• Oosterwegel M, van de Wetering M, Clevers H
• HMG box proteins in early T-cell differentiation.
• Thymus. 1993; 22: 67-81
• Display abstract

• The central theme of this review is the molecular basis for commitment of cells to the T-cell lineage. Principles of transcriptional regulation are illustrated by two examples; the role of GATA-1 during erythroid differentiation and the function of MyoD-like proteins in myogenesis. Several regulatory proteins have been described in the T-cell lineage. Here, we focus attention on the HMG box family of DNA binding proteins. This recently defined family can be divided in two subfamilies: the HMG/UBF and the TCF/SOX group. The first group contains at least two HMG boxes and binds DNA non-specifically, while the other group of proteins has one HMG box and interacts with DNA sequence-specifically. Characteristics of the most prominent members of both subfamilies will be discussed. In particular, we will address the role of HMG box proteins in controlling the expression of T-cell specific proteins during differentiation.

• Dooijes D, van de Wetering M, Knippels L, Clevers H
• The Schizosaccharomyces pombe mating-type gene mat-Mc encodes a sequence-specific DNA-binding high mobility group box protein.
• J Biol Chem. 1993; 268: 24813-7
• Display abstract

• The Schizosaccharomyces pombe gene mat-Mc plays a determinative role in the sexual differentiation of the fission yeast. The mat-Mc protein has been suggested to belong to a novel family of so-called high mobility group (HMG) box proteins, characterized by homology to high mobility group-1 and -2 proteins. Several HMG box proteins, including the mammalian sex-determining gene product SRY and the lymphoid transcription factors TCF-1 and LEF-1, have been shown to bind to DNA in a sequence-specific fashion. To analyze possible DNA-binding properties of mat-Mc, we have cloned and expressed its putative HMG box in Escherichia coli. Gel retardation analysis revealed that the mat-Mc HMG box recognizes the AACAAAG heptamer in a sequence-specific fashion. Combined T-->C and A-->I substitutions on both strands of the AACAAAG heptamer, which change the surface of the major groove while leaving the minor groove intact, did not interfere with sequence-specific binding of mat-Mc. Methylation interference analysis confirmed that the mat-Mc HMG box contacts adenine residues in the minor groove. By using a circular permutation assay, the mat-Mc HMG box was observed to bend DNA. These results indicate that mat-Mc is indeed a member of the HMG box family with DNA-binding characteristics assigned earlier to other members of this novel transcription factor family.

• Goze C, Poulat F, Berta P
• Partial cloning of SOX-11 and SOX-12, two new human SOX genes.
• Nucleic Acids Res. 1993; 21: 2943-2943

• Bruhn SL, Housman DE, Lippard SJ
• Isolation and characterization of cDNA clones encoding the Drosophila homolog of the HMG-box SSRP family that recognizes specific DNA structures.
• Nucleic Acids Res. 1993; 21: 1643-6
• Display abstract

• Recently an HMG-box protein denoted SSRP1, for structure-specific recognition protein 1, has been discovered which binds to specific DNA structural elements such as the bent, unwound conformations that occur upon the formation of intrastrand crosslinks by the anticancer drug cisplatin. The SSRP family includes the mouse protein T160, which recognizes recombination signal sequences. In order to delineate functional domains more clearly, a homolog of SSRP1 was cloned from Drosophila melanogaster. This homolog maps to polytene region 60A (1-4) and shares 54% identity with human SSRP1. Comparison of the predicted amino acid sequences among SSRP family members reveals 48% identity, with structural conservation in the carboxy terminus of the HMG box as well as domains of highly charged residues. Interestingly, however, the most highly conserved regions of the protein are in the less well understood amino terminus, strongly suggesting that this portion of the protein is critical for its function.

• Giese K, Cox J, Grosschedl R
• The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures.
• Cell. 1992; 69: 185-95
• Display abstract

• The high mobility group (HMG) domain is a DNA-binding motif that is associated with several eukaryotic regulatory proteins, including the lymphoid enhancer-binding factor LEF-1 and the testis-determining factor SRY. Here, we provide evidence that DNA binding by the HMG domain of LEF-1 involves primarily minor groove contacts and induces a bend of approximately 130 degrees in the DNA helix. Bending was also found to accompany sequence-specific DNA binding by the SRY-HMG domain. Examining possible regulatory roles of HMG domain-induced DNA bends, we found that LEF-1 can function in a manner similar to bacterial integration host factor and facilitate communication between widely separated protein-binding sites in a recombination assay. Together with the previous observation that LEF-1 by itself is unable to augment basal promoter activity, these data suggest that HMG domain proteins can serve as "architectural" elements in the assembly of higher-order nucleoprotein structures.

• Crippa MP, Alfonso PJ, Bustin M
• Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain.
• J Mol Biol. 1992; 228: 442-9
• Display abstract

• Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.

• Bianchi ME, Falciola L, Ferrari S, Lilley DM
• The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.
• EMBO J. 1992; 11: 1055-63
• Display abstract

• The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other. Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not. Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments. HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding. Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins. However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins.

• van de Wetering M, Clevers H
• Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.
• EMBO J. 1992; 11: 3039-44
• Display abstract

• The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

• Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi ME
• SRY, like HMG1, recognizes sharp angles in DNA.
• EMBO J. 1992; 11: 4497-506
• Display abstract

• HMG boxes are DNA binding domains present in chromatin proteins, general transcription factors for nucleolar and mitochondrial RNA polymerases, and gene- and tissue-specific transcriptional regulators. The HMG boxes of HMG1, an abundant component of chromatin, interact specifically with four-way junctions, DNA structures that are cross-shaped and contain angles of approximately 60 and 120 degrees between their arms. We show here also that the HMG box of SRY, the protein that determines the expression of male-specific genes in humans, recognizes four-way junction DNAs irrespective of their sequence. In addition, when SRY binds to linear duplex DNA containing its specific target AACAAAG, it produces a sharp bend. Therefore, the interaction between HMG boxes and DNA appears to be predominantly structure-specific. The production of the recognition of a kink in DNA can serve several distinct functions, such as the repair of DNA lesions, the folding of DNA segments with bound transcriptional factors into productive complexes or the wrapping of DNA in chromatin.

• Radic MZ, Saghbini M, Elton TS, Reeves R, Hamkalo BA
• Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA.
• Chromosoma. 1992; 101: 602-8
• Display abstract

• The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.

• Dalrymple BP, Peters JM
• Characterization of a cDNA clone from the haemoparasite Babesia bovis encoding a protein containing an "HMG-Box".
• Biochem Biophys Res Commun. 1992; 184: 31-5
• Display abstract

• The complete nucleotide sequence of a Babesia bovis cDNA clone encoding a protein containing an HMG-Box has been determined. The predicted protein of 97 amino acids has a molecular weight of 11,116. It exhibits approximately 45% overall amino acid identity with the Saccharomyces cerevisiae non-histone protein 6A (NHP6A) and approximately 57% identity in the HMG-Box. The B. bovis protein has been designated NHP1. Like HNP6A, and unlike most other HMG1 homologues, NHP1 does not have a basic or an acidic carboxy-terminal domain. The amino acid sequence of HNP1 is much less similar to HMG1 homologues of another protozoan, Tetrahymena thermophila, than to the HMG1 homologues identified in S. cerevisiae, plants and vertebrates. This suggests that the T. thermophila proteins may not be true HMG1 homologues, or that they may be evolving at a much faster rate.

• Giese K, Amsterdam A, Grosschedl R
• DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1.
• Genes Dev. 1991; 5: 2567-78
• Display abstract

• Lymphoid enhancer-binding factor 1 (LEF-1) is a pre-B and T lymphocyte-specific nuclear protein that participates in the regulation of the T-cell antigen receptor (TCR) alpha enhancer by binding to the nucleotide sequence 5'-CCTTTGAA. LEF-1 protein shares with the nonhistone high mobility group protein 1 (HMG-1) and several transcriptional regulators a single region of amino acid homology, termed the HMG box, which has been implicated in DNA binding. Here, we report the biochemical analysis of the interaction of this novel structural motif with DNA. First, amino- or carboxy-terminal truncations of the LEF-1 polypeptide delineated the HMG box as the DNA-binding domain. We purified to homogeneity a LEF-HMG domain peptide expressed in Escherichia coli and determined the equilibrium constant for specific binding to DNA as 1 x 10(-9) M. Second, cotranslation of wild-type and various truncated LEF-1 polypeptides did not generate any DNA-binding heterodimers, suggesting that LEF-1 can bind DNA as a monomer. Third, methylation interference analysis indicated that the HMG domain specifically contacts DNA on one side of the double helix. Finally, changes of amino acids that are conserved among various members of the family of HMG-box proteins decreased the affinity of DNA binding by one to three orders of magnitude. Together, these data define the characteristics of specific DNA-binding by the HMG domain of LEF-1.

• Fedorova NA
• [High mobility group proteins: structure, localization, function]
• Biokhimiia. 1991; 56: 9-18
• Display abstract

• High motility group proteins (HMG) are extracted by 5% HCIO4 and 0.35 M NaCl and are characterized by low molecular mass and a high content of acidic and basic amino acids. There is evidence that HMG are involved in the formation of transcriptionally active chromatin.

• Lund T, Berg K
• Metaphase-specific phosphorylations weaken the association between chromosomal proteins HMG 14 and 17, and DNA.
• FEBS Lett. 1991; 289: 113-6
• Display abstract

• The high-mobility-group proteins HMG 14 and 17 have been isolated from human cells arrested in metaphase. The affinity between an unphosphorylated and two phosphorylated forms of these proteins, and DNA has been investigated using columns of single-stranded and double-stranded DNA. It was shown that the most phosphorylated forms had much lower affinity for single-stranded and double-stranded DNA compared to the unphosphorylated form present in interphase cells. The results are in accordance with the view that HMG 14 and 17 may dissociate transiently from chromatin during mitosis.

• Bianchi ME
• Production of functional rat HMG1 protein in Escherichia coli.
• Gene. 1991; 104: 271-5
• Display abstract

• High-mobility group-1 protein (HMG1) was produced in Escherichia coli under the control of the T7 promoter/T7 RNA polymerase system. The protein can be produced and purified with yields similar to those obtained from animal tissues. HMG1 purified from E. coli is homogeneous and capable of selectively binding cruciform DNA, indicating that post-translational processing of vertebrate HMG1 is not necessary for its DNA-binding ability.

• Kolodrubetz D
• Consensus sequence for HMG1-like DNA binding domains.
• Nucleic Acids Res. 1990; 18: 5565-5565

• Bustin M, Lehn DA, Landsman D
• Structural features of the HMG chromosomal proteins and their genes.
• Biochim Biophys Acta. 1990; 1049: 231-43

• Christen T, Bischoff M, Hobi R, Kuenzle CC
• High mobility group proteins 1 and 2 bind preferentially to brominated poly(dG-dC).poly(dG-dC) in the Z-DNA conformation but not to other types of Z-DNA.
• FEBS Lett. 1990; 267: 139-41
• Display abstract

• Three proteins from bull testis, previously thought to be Z-DNA-binding proteins but recently found to recognize brominated poly(dG-dC). poly(dG-dC) by criteria different from the Z-conformation, were partially sequenced. Of these, the 31 kDa protein was identified as a member of the high mobility group 2 protein family, and the 33 kDa protein as a member of the high mobility group 1 protein family. Both proteins had molecular weights approximately 30% higher than expected, indicating considerable posttranslational modification. In contrast, the 58 kDa protein remained unidentified for lack of any significant homology with known protein sequences.

• Bianchi ME, Beltrame M, Paonessa G
• Specific recognition of cruciform DNA by nuclear protein HMG1.
• Science. 1989; 243: 1056-9
• Display abstract

• Cruciform DNA, a non-double helix form of DNA, can be generated as an intermediate in genetic recombination as well as from palindromic sequences under the effect of supercoiling. Eukaryotic cells are equipped with a DNA-binding protein that selectively recognizes cruciform DNA. Biochemical and immunological data showed that this protein is HMG1, an evolutionarily conserved, essential, and abundant component of the nucleus. The interaction with a ubiquitous protein points to a critical role for cruciform DNA conformations.

• Solomon MJ, Strauss F, Varshavsky A
• A mammalian high mobility group protein recognizes any stretch of six A.T base pairs in duplex DNA.
• Proc Natl Acad Sci U S A. 1986; 83: 1276-80
• Display abstract

• alpha-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey alpha-satellite DNA. We have used DNase I footprinting to identify 50 alpha-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. alpha-Protein binds with approximately equal affinity to any run of six or more A X T base pairs in duplex DNA, to many, if not all, runs of five A X T base pairs, and to a small number of other sequences within otherwise (A + T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, alpha-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, alpha-protein recognizes a configuration of the minor groove characteristic of short runs of A X T base pairs. We discuss possible functions of alpha-protein and the similarities in DNA recognition by alpha-protein and the antibiotic netropsin.

• Cary PD, Turner CH, Leung I, Mayes E, Crane-Robinson C
• Conformation and domain structure of the non-histone chromosomal proteins HMG 1 and 2. Domain interactions.
• Eur J Biochem. 1984; 143: 323-30
• Display abstract

• The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367-374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A + B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil----helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57--C-terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.

• Shik VV, Beliavskii AV, Mirzabekov AD, Ermekova VM, Beletskii IP
• [New immunochemical method of localizing proteins on DNA. The primary organization of nucleosomes containing proteins HMG 14 and 17]
• Dokl Akad Nauk SSSR. 1984; 279: 1254-6

• McGhee JD, Rau DC, Felsenfeld G
• The high mobility group proteins HMG 14 and 17, do not prevent the formation of chromatin higher order structure.
• Nucleic Acids Res. 1982; 10: 2007-16
• Display abstract

• The high mobility group proteins, HMG 14 and 17, have been associated with the chromatin of active genes (refs 1-8), although how they function is not known. We use sedimentation and electric dichroism to investigate the effect of HMG 14 and 17 on the condensation of chicken erythrocyte chromatin into higher order structure. We find no evidence that excess HMG 14 and 17 induce an extended configuration, either in bulk chromatin or in the chromatin of the chicken beta-globulin gene.

• Goodwin GH, Brown E, Walker JM, Johns EW
• The isolation of three new high mobility group nuclear proteins.
• Biochim Biophys Acta. 1980; 623: 329-38
• Display abstract

• In addition to the four high mobility group non-histone chromosomal proteins HMG 1, 2, 14 and 17 and histone H1, perchloric acid extracts of nuclei contain a number of other smaller low molecular weight proteins. Three of these proteins (HMG 18, 19A, and 19B) have been purified and characterized. Protein HMG 18 has high lysine and alanine contents, resembling histone H1. Proteins HMG 19A and 19B have high contents of basic and acidic amino acids and resemble HMG proteins 1, 2, 14 and 17. N-terminal sequence analyses of the proteins show that they are not degradation products of histones or the other HMG proteins. However, there are sequence similarities between HMG 18 and histone H5, and between HMG 19B and HMG 17, supporting the view that the HMG proteins and the lysine-rich histones are functionally related.

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