The HMG-box: a versatile protein domain occurring in a wide variety of DNA-binding proteins.

The HMG-box domain of approximately 75 amino acid residues was originally identified as the domain that mediates the DNA-binding of chromatin-associated high-mobility group (HMG) proteins of the HMGB type. In the last few years, HMG-box domains have been found in various DNA-binding proteins including transcription factors and subunits of chromatin-remodeling complexes. HMG-box domains mediate either non-sequence-specific (e.g., HMGB-type proteins) or sequence-specific (e.g., transcription factors) DNA binding. Both types of HMG-box domains bind non-B-type DNA structures (bent, kinked and unwound) with high affinity. In addition, HMG-box domains are involved in a variety of protein-protein interactions. Here, we have examined the human and plant genomes for genes encoding HMG-box domains. Compared to plants, human cells contain a larger variety of HMG-box proteins. Whereas in humans transcription factors are the most divergent group of HMG-box proteins, in plants the chromosomal HMGB-type proteins are most variable.

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.

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.

HMG1 protein stimulates DNA end joining by promoting association of DNA molecules via their ends.

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA-binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C-terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive-end and blunt-end DNA ligation by T4 DNA ligase via its B domain. The C-terminal flanking sequence of the B domain (seven basic residues out of approximately 18) and a number of conserved amino-acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull-down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation.

DNA bending by the chromosomal protein HMG1 and its high mobility group box domains. Effect of flanking sequences.

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.

Formation of large nucleoprotein complexes upon binding of the high-mobility-group (HMG) box B-domain of HMG1 protein to supercoiled DNA.

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.

Fludarabine triphosphate inhibits nucleotide excision repair of cisplatin-induced DNA adducts in vitro.

Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.

cDNA sequence and structure of a trout HMG-2 gene. Evidence for a trout-specific 3'-untranslated region.

A complete cDNA sequence (1026 bp) and a partial structure of a gene encoding the trout testis chromosomal HMG (high mobility group) 2 protein (HMG-T2) is presented. The deduced protein consists of 214 amino acids and shares over 80% similarity to the trout HMG-1 protein (HMG-T1) as well as to the mammalian or avian HMG-2 proteins. Northern blot analysis revealed two transcripts, a major one of 1.2 kb and a minor one of 1.6 kb. Southern analysis and polymerase chain reaction of trout genomic DNA indicated that the HMG-T2 gene is encoded by several introncontaining genes. The 5'-UTR (untranslated region) of the HMG-T2 is interrupted by an intron and the coding region of the HMG-T2 is divided into four exons by three relatively short introns (173, 91 and 78 bp). The exon/intron boundaries of trout HMG-2 are identical to those of human HMG-2, as reported earlier [Shirakawa and Yoshida, J. Biol. Chem. 267 (1992) 6641-6645], suggesting the evolution of the HMG-1/2 family genes from a common ancestor. Phylogenetic analysis indicated that the common ancestor of trout HMG-1/2 genes very likely diverged from the ancestor of the mammalian (or avian) HMG-1/2 genes before its separation into two distinct mammalian or (avian) HMG-1 and HMG-2 genes. Sequence comparisons of the 3'UTR of trout HMG-2 cDNA with the corresponding regions in the mammalian (or avian) HMG-2 revealed that the trout 3'-UTR was clearly distinct from the 3'-UTR of the mammalian or avian HMG-2 cDNAs which were otherwise remarkably well conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA sequence and structure of a gene encoding trout testis high-mobility-group-1 protein.

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high-mobility-group (HMG) proteins, HMG-T1 and HMG-T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG-1 protein (referred as to HMG-T1) has been determined. The deduced HMG-T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG-1 proteins. A single-sized mRNA for HMG-T1 has been detected by Northern-blot analysis consistent with the size derived from the HMG-T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG-1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG-1 sequences, intronless HMG-T1 sequences were not found in the fish genome. Southern-blot analysis suggested that the trout testis HMG-1 gene is encoded by at least two sequences with high similarity. A gene encoding HMG-T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp). The trout testis HMG-1 gene is organized into five exons (four exons corresponding to the protein-coding region) and its exon/intron boundaries are identical to those of the human HMG-2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641-6645] suggesting the evolution of HMG-1 and HMG-2 genes from a common ancestor.

Calcium binding to HMG1 protein induces DNA looping by the HMG-box domains.

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.

DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.

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.

A retropseudogene for non-histone chromosomal protein HMG-1.

Southern analysis of the human genome revealed that there are several sequences with homology to the nonhistone chromosomal protein HMG-1. The majority of the HMG-1 sequences are intronless as suggested from the polymerase chain reaction of HeLa DNA. Sequencing of a clone from a human placenta genomic library revealed that the clone was intronless and displayed 99% homology to the human HMG-1 cDNA. The 5' regulatory motif, CCAAT, is present in the clone but there is no TATA-box. Most of the differences between the HMG-1 cDNA sequence and the clone involve point mutations with no interruption of the reading frame. The sequence is flanked at 5' and 3' ends by a 15 nucleotide long direct repeat suggesting that the clone is a processed HMG-1 retropseudogene. Sequence differences between the reading frames of the HMG-1 pseudogene and HMG-1 cDNA indicated that the pseudogene arose relatively late in evolution, approximately one million years ago. The present paper is the first study on a genomic sequence related to HMG-1 genes.

Non-histone chromosomal protein HMG1 reduces the histone H5-induced changes in c.d. spectra of DNA: the acidic C-terminus of HMG1 is necessary for binding to H5.

Chemical cross-linking was used to study the interaction between non-histone high-mobility-group (HMG)1 and histone H5 in free solution. The presence of acidic C-terminal domain in HMG1 was shown to be a prerequisite for HMG1 binding to histone H5. The objective of this communication is to ascertain whether HMG1 could affect the conformation of DNA associated with a linker histone H5. Complexes of histone H5 with chicken erythrocyte DNA or an alternating purine-pyrimidine polynucleotide poly[d(A-T)] were prepared at different molar ratios H5/DNA. Changes in DNA conformation in the complexes with histone H5 or H5/HMG1 were monitored by circular dichroism (c.d.). Depending on the molar ratio H5/poly[d(A-T)], under conditions limiting the complex aggregation, three distinct types of c.d. spectra were observed. The addition of HMG1 to H5-DNA complexes reduced in all cases the histone H5-induced conformational changes in poly[d(A-T)]. The sensitivity of H5-poly[d(A-T)] complexes to HMG1 was inversely proportional to the amount of H5 in the complex. The effect of HMG1 was not observed upon removal of the acidic C-terminal domain of HMG1.

Calcium modulates the binding of high-mobility-group protein 1 to DNA.

Binding of 45Ca2+ to nonhistone protein HMG1 was detected after fixation of the protein to nitrocellulose membrane. The same experiment with HMG1 peptides, derived from HMG1 by protease V8 digestion, allowed to identify the highly glutamic and aspartic C-terminal domain of HMG1 as a 45Ca2(+)-binding region. Measurements of 32P-labeled DNA retention on nitrocellulose filters revealed that in the absence of Ca2+, the affinity of HMG1 for linear DNA decreased upon an increase of pH from 7 to 8.4. However, when Ca2+ was included in the assay buffer, the affinity of HMG1 for DNA remained unchanged between pH 7 to 8.4 and was higher than in the absence of Ca2+. The effect of Ca2+ on HMG1 - DNA interaction was no longer observed upon removal of the C-terminal domain from HMG1.

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2.

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.

Binding of non-histone chromosomal protein HMG1 to histone H3 in nucleosomes detected by photochemical cross-linking.

The interaction of non-histone chromosomal protein HMG1 with core histones in nucleosomes was studied via reconstitution and photochemical cross-linking. The results obtained indicated that photoaffinity-labeled HMG1 interacted in nucleosomes with histone H3. Similar experiments with peptides derived from HMG1 by V8 protease digestion allowed to identify N-terminal domain of HMG1 (peptide V3) as a binding region for histone H3 in nucleosomes.

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking.

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.

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 approximately 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of approximately 145 base pairs.

Histone H1 in nuclei of butyrate-treated murine lymphosarcoma cells has increased affinity for heparin.

Nuclei from butyrate-treated murine lymphosarcoma cells were incubated with different amounts of the polyanion heparin, which is known to interact predominantly with chromatin-associated histones. Unlike isolated histone H1, histone H1 in the nuclei of butyrate-treated cells was found to display an enhanced affinity for the binding to heparin as compared to histone H1 from control cells. Dephosphorylation of histone H1 as a result of butyrate treatment of the cells is discussed as a possible factor involved in the observed higher affinity of the protein for heparin.