Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.     

Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.     

Nature of full-length HMGB1 binding to cisplatin-modified DNA

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.     

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

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.     

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.

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.     

HMGB binding to DNA: single and double box motifs

High mobility group (HMG) proteins are nuclear proteins believed to significantly affect DNA interactions by altering nucleic acid flexibility. Group B (HMGB) proteins contain HMG box domains known to bind to the DNA minor groove without sequence specificity, slightly intercalating base pairs and inducing a strong bend in the DNA helical axis. A dual-beam optical tweezers system is used to extend double-stranded DNA (dsDNA) in the absence as well as presence of a single box derivative of human HMGB2 [HMGB2(box A)] and a double box derivative of rat HMGB1 [HMGB1(box A+box B)]. The single box domain is observed to reduce the persistence length of the double helix, generating sharp DNA bends with an average bending angle of 99 ± 9° and, at very high concentrations, stabilizing dsDNA against denaturation. The double box protein contains two consecutive HMG box domains joined by a flexible tether. This protein also reduces the DNA persistence length, induces an average bending angle of 77 ± 7°, and stabilizes dsDNA at significantly lower concentrations. These results suggest that single and double box proteins increase DNA flexibility and stability, albeit both effects are achieved at much lower protein concentrations for the double box. In addition, at low concentrations, the single box protein can alter DNA flexibility without stabilizing dsDNA, whereas stabilization at higher concentrations is likely achieved through a cooperative binding mode.     

Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.     

Interaction with p53 enhances binding of cisplatin-modified DNA by high mobility group 1 protein

A nonhistone chromosomal protein, high mobility group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds preferentially to cisplatin-modified DNA. HMG1 also functions as a coactivator of p53, a tumor suppressor protein. We investigated physical interactions between HMG1 and p53 and the influence of p53 on the ability of HMG1 to recognize damaged DNA. Using immunochemical coprecipitation, we observed binding of HMG1 and p53. Interaction between HMG1 and p53 required the HMG A box of HMG1 and amino acids 363-376 of p53. Cisplatin-modified DNA binding by HMG1 was significantly enhanced by p53. An HMG1-specific antibody that recognized the A box of this protein also stimulated cisplatin-modified DNA binding. These data suggest that an interaction with either p53 or antibody may induce conformational change in the HMG1 A box that optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and p53-mediated DNA repair.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Physicochemical properties of a novel Mr-21 000 Ca2+-binding protein of bovine brain.

The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% alpha-helix, 18% beta-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% alpha-helix, 18% beta-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.