Hierarchy of binding sites for chromosomal proteins HMG 1 and 2 in supercoiled deoxyribonucleic acid

The interaction of chromosomal proteins HMG 1 and 2 with various DNA structures has been examined with plasmid pPst-0.9, which contains DNA sequences that can form the Z-DNA conformation and palindromic sequences that can form cruciform structures. Direct binding and competition experiments with 32P-labeled plasmid indicated that proteins HMG 1 and 2 preferentially bind to supercoiled form I DNA as compared to double-stranded linear DNA. The preferential binding to form I is due to the presence of single-stranded regions in this DNA. The binding of HMG 1 and 2 to the form I plasmid results in inhibition of S1 nuclease digestion in a selective manner. The B-Z junction is preferentially protected as compared to the cruciform, which in turn is more protected than other minor S1-sensitive structures present in pPst-0.9. Our results indicate that the binding of HMG 1 and 2 proteins to DNA is not random in that HMG 1 and 2 can distinguish between various S1 nuclease sensitive sites in the plasmid. The existence of a hierarchy of DNA binding sites for these proteins suggests that they can selectively affect the structure of distinct regions in the genome.     

Nonhistone proteins HMG1 and HMG2 suppress the nucleosome assembly at physiological ionic strength

The effect of nonhistone protein HMG1 and HMG2 from pig thymus on the in vitro nucleosome assembly has been examined with plasmid pSV2-gpt DNA and pig thymus core histones in the presence of DNA topoisomerase I. In the absence of core histones, the direct binding of HMG proteins could induce negative superhelical turns in DNA at low ionic strength, but not at physiological ionic strength. The nucleosome formation in the higher histone-to-DNA ratios at physiological ionic strength was not facilitated by HMG proteins, in contrast to poly(L-glutamic acid). HMG proteins suppressed the nucleosome assembly in the moderate histone-to-DNA ratios, resulting in the reduction of fully supercoiled DNA topoisomers. The suppression by HMG proteins was not cancelled by poly(L-glutamic acid). These suggest that the highly acidic carboxy terminal of HMG proteins does not act as an assembly factor, and that the HMG proteins, on the contrary, suppress the nucleosome formation, probably by binding to DNA in a way to inhibit the assembly into core particles.     

Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific β-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.     

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.     

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.     

HMG box A in HMG2 protein functions as a mediator of DNA structural alteration together with box B

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.     

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.     

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.     

High affinity binding of proteins HMG1 and HMG2 to semicatenated DNA loops

Background  

Proteins HMG1 and HMG2 are two of the most abundant non histone proteins in the nucleus of mammalian cells, and contain a domain of homology with many proteins implicated in the control of development, such as the sex-determination factor Sry and the Sox family of proteins. In vitro studies of interactions of HMG1/2 with DNA have shown that these proteins can bind to many unusual DNA structures, in particular to four-way junctions, with binding affinities of 10⁷ to 10⁹ M^-1.     

Interaction of high mobility group proteins HMG 1 and HMG 2 with nucleosomes studied by gel electrophoresis

The binding of isolated high mobility group proteins HMG (1+2) with nucleosomes was studied using gel electrophoresis. The interaction of HMG (1+2) with mononucleosomes could be detected as a new discrete electrophoretic band with a decreased mobility only after cross-linking of HMG (1+2)-nucleosome complex by formaldehyde. Approximately two molecules of the large HMG proteins were bound per nucleosomal particle of a DNA length of 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of 145 base pairs.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of the sequence non-specific DNA binding HMG1/2-box family of proteins, has been examined by both mutation analyses and molecular modeling techniques. Substitution of the residue 102F, which is characteristically exposed to solvent, with a small hydrophobic amino acid affected its DNA binding activity. However, no additional effect was observed by the further mutation of flanking 101F. Molecular dynamics simulation and modeling studies revealed that 102F intercalates into DNA base-pairs, being supported by the flanking 101F. The mutants with a small hydrophobic residue at position 102 tolerated the substitution for 101F because the side chain at position 102 is too short to intercalate. Thus the intercalation of 102F and the positive effect of the flanking 101F residue are important for the sequence non-specific DNA binding of the HMG1/2-box. The conserved basic residues of 95K, 96R and 109R were also examined for their roles in DNA binding. These residues interacted with DNA mainly by electrostatic interaction and maintained the location of the box on the DNA, which prescribed the intercalation of 102F. The DNA intercalation by HMG1 consists of an ingenious mechanism which brings DNA conformational changes necessary for biological functions.     

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.     

HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.     

Structural requirements for cooperative binding of HMG1 to DNA minicircles

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.