Immunochemical analysis of the exposure of high mobility group protein 14 and 17 surfaces in chromatin

Antisera were elicited against synthetic peptides corresponding either to regions common to all members of the high mobility group 14 and 17 protein family protein or to distinct domains of the HMG-14 or HMG-17 subgroup. The antisera were used to probe the accessibility of various HMG domains in chromatin. Competitive enzyme-linked immunosorbent assays indicate that the central region of the proteins, which contains their DNA binding domain and is positively charged, is exposed to a smaller degree than the C-terminal region of the proteins, which has a net negative charge. The C-terminal regions of the HMG-14 and HMG-17 proteins are exposed and available to interact with other proteins.     

Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2

Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin. One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested. In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions. The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish. Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a. One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes. In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity. The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro. Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.     

Mechanism for specificity by HMG-1 in enhanceosome assembly

Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.     

The binding of the chromosomal protein HMG-2a to DNA regions of reduced stabilities

The interaction of immunopurified high mobility group 2a protein (HMG-2a) with DNA was examined by the nitrocellulose filter binding assay. The relative binding activity of HMG-2a for synthetic polynucleotides was: (dI).(dC) greater than (dA-dT).(dA-dT) greater than (dA).(dT) much greater than (dG).(dC) greater than (dG-dC).(dG-dC). The protein also exhibited a marked preference for (A + T)-rich restriction fragments derived from rat and Drosophila satellites, yeast centromeres, phage lambda, and the ovalbumin gene and its 5' flanking sequences. These preferential DNA interactions occurred at ionic strengths and temperatures within the physiological range which argue for an in vivo role of DNA stability in dictating the genomic distribution of the large Mr HMG proteins.     

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

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.     

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

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.     

The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly

HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.     

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

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.