Structure of the HMG box motif in the B-domain of HMG1.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.     

Interactions between HMG boxes.

Many proteins consist of subdomains that can fold and function independently. We investigate here the interaction between the two high mobility group (HMG) box subdomains of the nuclear protein rHMG1. An HMG box is a conserved amino acid sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains that is active in binding DNA in a sequence or structure-specific manner. In the case of HMG1, each box can bind structural DNA substrates including four-way junctions (4WJs) and branched or kinked DNA duplexes. Since proteins containing up to six HMG boxes are known, the question arises whether linking subdomains together influences the folding or function of individual boxes. In an effort to understand interactions between individual DNA-binding domains in HMG1, we created new fusion proteins: one is an inversion of the order of the AB di-domain in HMG1 (BA); in the second, we added a third A domain C-terminal to the AB di-domain (ABA). Pairs of boxes, AB or BA, behave similarly and are functionally active. By contrast, the ABA triple subdomain construct is partially unfolded and is less active than individual boxes or di-domains. Thus, long-range inter-domain effects can influence the activity of HMG boxes.     

Unwinding of DNA by nonhistone protein HMG1 and HMG2

The enzyme kinetic studies with endonucleases specific for single-stranded DNA and the thermal denaturation analyses of DNA showed that a high mobility group (HMG) nonhistone protein fraction HMG (1 + 2), composed of HMG1 and HMG2, has an activity to unwind DNA partially at low protein-to-DNA weight ratio. Isolated HMG1 and HMG2 have the same activity. Divalent cations such as Mg++ or Ca++ were necessary for the unwinding reaction. A peptide containing high glutamic and aspartic (HGA) region, isolated from the tryptic digest of HMG (1 + 2), unwound DNA depending on the presence of Mg++ or Ca++, suggesting that the HMA region in HMG protein is the active site for the DNA unwinding reaction. Poly-L-glutamic acid, employed as a model peptide of the HGA region, showed the activity. Finally, mechanisms of the DNA unwinding reaction by the HMG protein and possible role of the divalent cations are discussed.     

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.     

DNA binding by single HMG box model proteins

The HMG1/2 family is a large group of proteins that share a conserved sequence of ~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 Mg²⁺. 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 Mg²⁺ is present in the nucleus and these proteins are expressed at a very high level.     

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.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

Unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined with endonuclease

A high mobility group (HMG) nonhistone protein fraction HMG(1 + 2), composed of HMG1 and HMG2, was prepared from pig thymus chromatin. In order to examine a possibility that the HMG(1 + 2) participates in the unwinding of the DNA double-helix, DNA hydrolysis assay systems with the endonucleases specific for single-stranded DNA were employed. In the presence of HMG(1 + 2), the hydrolysis of double-stranded DNA by N. crassa endonuclease was markedly promoted, while the hydrolysis of single-stranded DNA was hardly enhanced. The reaction kinetic data showed that the stimulation of the hydrolysis of double-stranded DNA in the presence of HMG(1 + 2) was due to the unwinding of the DNA double-helix by the HMG(1 + 2), and not due to stimulation of enzyme activity of the endonuclease by the protein. The unwinding reactions were dependent on the HMG protein concentration at low weight protein to DNA ratios and reached a maximum at the ratio of 0.025. The region unwound in the whole DNA was partial. Similar results were obtained for experiments with nuclease S1. Isolated HMG1 and HMG2 fractions showed DNA unwinding activity of similar extents. The association constant obtained by fluorescence quenching analysis showed that the HMG(1 + 2) has higher affinity to single-stranded DNA than to double-stranded DNA. The susceptibility to the unwinding differed with the DNA source. These results suggest that HMG(1 + 2) at a low weight protein to DNA ratio binds to some limited double-stranded region in DNA and unwinds the DNA partially.     

Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.     

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.