From the Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
High mobility group (HMG)1 proteins 1 and 2 are relatively abundant chromosomal proteins with two homologous DNA-binding domains ("HMG boxes"), A and B, each of ~80-90 amino acid residues, linked by a short basic region to the C-terminal domain (reviewed in Refs. 1 and 2). The C-terminal domain of HMG1 is negatively charged with ~30 consecutive aspartate or glutamate residues and may be involved in interactions with other proteins such as histones, as well as in regulating DNA-binding affinity of the HMG1/2 proteins (3-8). Sequences with homology to the HMG boxes of HMG1 or HMG2 have been discovered in more than 100 DNA-binding proteins or transcription factors (for review, see Ref. 9).
HMG1 and its central B-domain (but not the N-terminal A-domain) can functionally substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome assembly during recombinational DNA inversion reaction in vitro (10), a process requiring DNA bending and looping. HMG1 could enhance progesterone receptor binding to its target DNA sequences by bending the DNA (11).
Although the exact cellular role of the HMG1 or HMG1 box proteins is not clear, the proteins have been implicated in a number of biologically fundamental processes including transcription, replication, and recombination (for review, see Ref. 2), where they may play an architectural role as DNA chaperones, which facilitate assembly of nucleoprotein higher order structures by bending or looping DNA or by stabilizing underwound DNA (6, 12-14, 26). In addition to the ability of HMG1 to act as a general, non-sequence-specific DNA bending/looping/wrapping protein, HMG1 and its HMG box domains are also able to recognize and bind altered (prebent and unwound) DNA conformations, as has been demonstrated with synthetic four-way junctions and adducts of the anticancer drug cisplatin on DNA (15-17).
Reports from several laboratories indicate that the precise length of the expressed HMG box polypeptides may be important for their DNA binding and bending activities. Extensions beyond the minimal HMG box in its natural sequence context increase DNA bend angle and binding affinity of the sequence-specific HMG box, hLEF-1 (18, 19). The addition of 19 extra amino acids to the C terminus of the Chironomus HMG1-like protein (cHMG1) results in an increased affinity for linear or four-way junction DNA (20). Comparison of HMG1 B-domain polypeptides of two different lengths indicates that the polypeptide with longer flanking sequences exhibits enhanced DNA binding and supercoiling, and promotes DNA bending and the self-association (oligomerization) of the DNA-bound HMG box (21). However, the exact contribution of the basic linker regions between the A/B- and B/C-domains of HMG1 for DNA bending activities of its HMG box domains has not been clear. In addition, conflicting results have been reported regarding the ability of the HMG1 A-domain to bend DNA (10, 11, 21, 22).
In the present work, we have used ligase-mediated circularization assays to investigate the effect of the acidic C-domain of HMG1 and the contribution of the HMG1 box A- and B-domains and their flanking sequences to the ability of the protein to bend DNA.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- The oligodeoxyribonucleotides were synthesized by the Integrated DNA Technologies (Coralville, IA) protocol and used directly for PCR. Promega and BioLabs restriction enzymes, T4 DNA kinase, T4 DNA ligase, T4 DNA polymerase, and Klenow fragment of DNA polymerase I were used. Isolation of plasmids and DNA fragments from 1% agarose gels was carried out using QiaPrep Spin Miniprep and gel purification kits (Qiagen), respectively. The radioactive labels were from Amersham Pharmacia Biotech.
Plasmids-- All HMG sequences were derived from rat HMG1 cDNA. Plasmid encoding the rat HMG1 protein was kindly provided by M. E. Bianchi (plasmid pT7-RNHMG1).
The HMG1 (A+B)-bidomain (residues 1-180), HMG box A (residues 1-84), and HMG box A' (residues 1-78) were prepared by PCR of the plasmid pT7-RNHMG1 using a sense primer 5'-GGATCCGGCAAAGGAGATCCTAAGAAG-3' (BamHI site is in italics) and antisense primers Brev (5'-GCAGGTCGACCTACTTCTCAGCCTTGACCAC-3') for the HMG1 (A+B)-bidomain, A (5'-GTCGACCTACTCCCCTTTGGGGGGGATG-3') for HMG box A and A' (5'-GTCGACCTAGTAGGTTTTCATTTC-3') for HMG box A' (SalI site is in italics, and the termination codon is underlined). The B-domains of different lengths were prepared by PCR of the plasmid pT7-RNHMG1 using an antisense primer, Brev, and the sense primers B (5'-GGATCCCCCAATGCCCCCAAG-3') for HMG box B (residues 92-180), B1 (5'-GGATCCGACCCCAATCCCCC-3') for HMG box B1 (residues 91-180), B2 (5'-GGATCCAAGGACCCCAATGCC-3') for HMG box B2 (residues 90-180), B3 (5'-GGATCCTTCAAGGACCCCAAT-3') for HMG box B3 (residues 89-180), B6 (5'-GGATCCAAAAAGAAGTTCAAG-3') for HMG box B6 (residues 86-180), and B7 (5'-GGATCCACCAAAAAGAAGTTC-3') for HMG box B7 (residues 85-180) (BamHI site is in italics). The antisense primer Brev(min) (5'-GTCGACCTAGTAGGCAGCAATATC-3') and either sense primer B or B7 were used for the PCR amplification of the "minimal" HMG box B (HMG box Bmin; residues 92-162) and the HMG box B' (residues 85-162), respectively (SalI site is in italics, and the termination codon is underlined). The gel-purified PCR products were cloned into a pGEM-T vector (Promega), and the positive clones were double-digested with BamHI and SalI restriction endonucleases to excise cDNAs encoding HMG polypeptides. The BamHI/SalI fragments were then cloned between the BamHI/SalI sites of the glutathione S-transferase (GST) fusion expression vector pGEX-4T1 (Amersham Pharmacia Biotech) to construct plasmids pGXRHMG1-(A+B), pGXRHMG1-A, pGXRHMG1-A', pGXRHMG1-Bmin, pGXRHMG1-B', pGXRHMG1-B, and pGXRHMG1-(B1-B7). The identity of the cloned HMG box sequences in the latter plasmids was verified by sequencing both strands using the dideoxynucleotide chain-termination method (T7 sequencing kit, Amersham Pharmacia Biotech).Overexpression and Purification of HMG1, HMG1 (A+B)-Bidomain, and
HMG Boxes from Escherichia coli--
HMG1 (A+B) and isolated HMG
boxes, encoded by plasmids pGXRHMG1-(A+B), pGXRHMG1-A, pGXRHMG1-A',
pGXRHMG1-Bmin, pGXRHMG1-B', pGXRHMG1-B, and
pGXRHMG1-(B1-B7) were expressed in BL 21 cells (Novagene) as fusion
proteins with GST to allow efficient synthesis and stability in
E. coli. The expressed HMG box-GST fused proteins were
selectively retained on glutathionine-Sepharose 4B columns. The columns
were washed thoroughly in 1× phosphate-buffered saline buffer, and the
HMG box polypeptides were then released from the column by cleavage
with thrombin, resulting in the HMG box polypeptides containing extra
N-terminal Gly-Ser dipeptides originating from the BamHI
site. This was followed by FPLC purification on a MonoS column (7). For
control experiments, two HMG1 B-domain polypeptides (residues 89-176
and 92-176) were expressed directly (unfused) under the control of the
T7 promoter/T7 RNA polymerase system as described previously (23). HMG1
protein was isolated either from BL 21 (DE3) cells harboring plasmid
pT7-RNHMG1 or from calf thymus and purified under nondenaturing
conditions as described previously (4, 7). Finally, purified HMG
proteins were dialyzed against 10 mM Tris-HCl, pH 7.6, 50 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol. The concentrations of the HMG1 and HMG1 (A+B)-bidomain were determined from the absorbance at 280 nm (4, 6). The concentrations of HMG boxes were determined by a Bio-Rad protein Coomassie G-250 dye assay and from the absorbance at 280 nm using = 12,500 M
1 cm
1 (24). The purity
and amounts of the HMG proteins were also checked by resolution of the
samples on 10% SDS-polyacrylamide slab gels in Tris-Tricine buffer at
50 V for 16 h, followed by Coomassie Blue G-250 staining (25).
DNA Probes--
The 123-bp DNA fragment was prepared by
AvaI digestion of the commercially available 123-bp ladder
(Life Technologies, Inc.). The 123-bp DNA fragment was labeled at its
5'-termini with [-32P]ATP using T4 polynucleotide
kinase and unincorporated label was removed on Sephadex G-50
minicolumns (Amersham Pharmacia Biotech).
Ligase-mediated Circularization Assay--
The circularization
assay used in this paper was based on existing protocols (10, 26) with
some modifications. In the standard protocol, 32P-labeled
123-bp DNA fragment (~1 nM) with cohesive
(AvaI) ends was preincubated on ice for 20 min with HMG1,
HMG1 (A+B)-bidomain, or isolated HMG box polypeptides (0.05-1.5
µM) in 30 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM dithiothreitol, and
0.5 mM ATP (1× T4 DNA ligase buffer) in a final volume of
10 µl. The DNA was then ligated with T4 DNA ligase (0.05 unit/reaction; Promega) at 30 °C for 5-40 min, followed by
termination at 65 °C for 15 min. Some of the reaction mixtures were
treated after termination of the ligation with 32 units of exonuclease
III (Promega) at 37 °C for 30 min. Before electrophoresis, all
samples were digested with proteinase K (1 µg/reaction) in 10 mM Tris-HCl, pH 8.0, 1% SDS, 4% sucrose, 0.025% each
xylene cyanol and bromphenol blue (electrophoretic loading buffer) at
37 °C for 60 min. They were loaded on prerun 5% polyacrylamide slab
gels (29:1 acrylamide/N,N'-methylene bisacrylamide) in 45 mM Tris base, 45 mM boric acid, 1.25 mM EDTA, pH 8.3-8.4 (0.5× TBE buffer). The gels were run
at 250 V for ~1.5 h (room temperature). On completion of
electrophoresis, the gels were vacuum-dried and either autoradiographed
at 70 °C (Agfa CP-B x-ray film) or visualized by phosphorimaging.
The amounts of monomer DNA circles were determined from quantitative
analyses of dried gels on a Molecular Dynamics Storm PhosphorImager
using the ImageQuant software.
Determination of the Binding Affinities of the HMG Boxes with DNA-- The Kd(app) was estimated from the gel mobility shift assay as the HMG protein concentration at the point in the titration where half of the input DNA had been complexed with protein, i.e. protein concentration at which 50% of the DNA was shifted (27, 28). DNA and proteins were mixed in 1× T4 DNA ligase buffer containing 0.05% Nonidet P-40, 0.2 mg/ml bovine serum albumin, and 6% glycerol as a DNA binding buffer (16). Briefly, 32P-labeled 123-bp DNA fragment (~0.1 nM) was incubated on ice with different amounts of HMG proteins (in a molar excess of DNA), and the complexes were resolved on prerun 5% polyacrylamide gels as indicated for the cyclization assay, but at 4 °C. Quantification was performed on a Molecular Dynamics Storm PhosphorImager.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the most direct methods in analyzing the DNA bending of non-sequence-specific proteins is the ligase-mediated circularization assay (DNA ring closure assay). This assay measures the efficiency with which T4-DNA ligase forms circles from fragments of DNA that are shorter than ~150 bp. In the absence of internal curvature, the stiffness of a short DNA fragment (<150 bp) prevents intramolecular alignment of its ends so that circles are detected only in the presence of a protein that bends DNA. In the present work, we have used the circularization assay to investigate the contribution of the HMG1 box A- and B-domains to, as well as the influence of the basic linker regions between A/B- and B/C-domains on, the previously reported ability of the HMG1 protein to bend DNA (10, 26). We also investigated whether the acidic C-terminal domain of HMG1 could modulate the DNA bending potential of the protein. The DNA bending analysis presented in this paper should be regarded as a qualitative approach rather than an attempt to determine explicitly the extent of DNA bending, which is to be estimated only by measuring true cyclization probabilities from cyclization kinetics or by mixed ligation (as defined in Refs. 30-32; see also "Discussion").
Protein sequences of rat HMG1, HMG1 lacking the acidic C terminus (referred to as HMG1 (A+B)-bidomain), and isolated HMG1 box A- and B-domains with flanking sequences of different lengths are shown in Fig. 1. HMG1 was expressed in E. coli as an unfused protein (encoded by plasmid pT7-RNHMG1), while the HMG polypeptides were synthetized in E. coli as GST fusion proteins to increase their stability in the bacterial cell. The GST-protein tag was subsequently removed and the released HMG polypeptides, having extra glycine and serine amino acids at their N termini contributed by the parental GST vector, were purified to homogeneity by FPLC chromatography (Fig. 2; some B-domain polypeptides were also expressed without the N-terminal Gly-Ser dipeptide and used as controls in DNA bending experiments; see "Discussion"). The ability of the expressed HMG polypeptides to bind DNA was verified in a gel retardation assay using labeled synthetic four-stranded Holliday junctions (four-way junction DNA) or linear 123-bp double-stranded DNA. The results obtained indicated that all of the HMG constructs could bind four-way junctions2 or the linear DNA used in the cyclization assay. The relative affinities of the HMG constructs for the linear 123-bp double-stranded DNA are presented as approximate Kd(app) values in Table I.
|
|
|
DNA Bending by HMG1 and HMG1 Lacking the Acidic C Terminus-- The ligase-mediated cyclization assay was performed at low DNA concentration (~1 nM) to reduce the formation of linear multimers of the 123-bp DNA probe in the control experiments, which used T4 DNA ligase alone. Production of DNA circles due to HMG-induced DNA bending was verified by their resistance to exonuclease III, which digests only linear DNA molecules. Extensive treatment of the ligated products with exonuclease III was necessary in order to completely digest linear DNA multimers, which in some cases resulted in a higher background. Using this approach, we have studied the effect of removal of the acidic C terminus of HMG1 on the ability of the protein to bend DNA in the ligase-mediated cyclization assay. As shown in Fig. 3, both HMG1 and its truncated form without the acidic C terminus (HMG1 (A+B)-bidomain) could bend DNA, indicating that the HMG box domains of the HMG1 protein are responsible for distortion of the DNA helix by bending as reported earlier (10, 11, 21, 26). The formation of DNA circles by HMG1 was relatively independent of the amount of the protein within the concentration range studied (0.05-1.5 µM; Fig. 3). On the other hand, removal of the acidic C terminus from HMG1 had a significant impact on the bending ability of HMG1. Whereas at lower HMG-to-DNA molar ratios HMG1 (A+B)-bidomain seemed to be more efficient than HMG1 in formation of DNA circles, at higher HMG1 (A+B)-to-DNA ratios, formation was markedly reduced relative to the full-length HMG1 protein (Fig. 3). These results might suggest that the acidic C terminus of HMG1 could modulate the ability of the native protein to bend DNA. We estimated Kd(app), the apparent Kd for the equimolar HMG-to-DNA interaction, for both HMG1 and HMG1 (A+B)-bidomain to be in the range of ~10-15 nM. Our Kd(app) value for HMG1 was in good agreement with the Kd(app) ~2-20 nM reported in Ref. 28 for HMG1 binding to 207-bp linear DNA in a similar DNA binding buffer. These results indicate that, at least under the conditions of the cyclization assay, removal of the acidic C-domain from HMG1 does not significantly change the DNA binding affinity of the protein.
|
|
|
|
The Involvement of N- and C-terminal Flanking Sequences of the HMG1 B-domain in DNA Bending-- Reports from several laboratories indicate that the precise length of the expressed sequence- or non-sequence-specific HMG box polypeptides may be important for their DNA binding and bending activities (18, 19, 21). However, the exact contribution of each of the two HMG1 boxes, A and B, to DNA bending is not clear. It is also not clear whether one or both of the N- and C-terminal flanking sequences of the B-domain are necessary for DNA bending activities of the HMG box. Initial experiments in this paper were aimed therefore at investigation of the effect of the B-domain's N- and/or C-terminal flanking sequences, in their natural sequence context within the native HMG1 protein, on its ability to bend DNA. As shown in Fig. 4, the minimal B-domain (referred to as the HMG box Bmin, residues 92-162, and defined as beginning 10 amino acids before the start of helix I and finishing at the end of helix III; see Ref. 1 and also Fig. 1 in this paper) did not bend DNA in a ligase-mediated circularization assay. Similarly, no DNA bending was observed with the minimal B-domain containing a 7-residue N-terminal extension (85TKKKFKD91), which lies in the linker region between the A/B-domains of HMG1 (HMG box B', residues 85-162 in Fig. 1). The ability of the HMG boxes Bmin and B' to bind DNA was verified with synthetic four-way junctions2 as well as with the linear DNA used in the circularization assay (the approximate Kd(app) values of the HMG boxes Bmin and B' with linear DNA were >200 nM; Table I). DNA ring closure assay with a minimal B-domain having an 18-amino acid addition at the C terminus, representing the linker region between B- and C-domains of HMG1, resulted in very weak DNA circle formation (Fig. 4; HMG box B, residues 92-180). However, similar experiments with a minimal B-domain, which had both 7-residue N-terminal and 18-residue C-terminal extensions (HMG box B7, residues 85-180), resulted in strong distortion of DNA by bending as revealed by markedly enhanced (~25-30-fold) formation of DNA circles relative to the B-domain lacking the N-terminal extension (Fig. 4). The estimated Kd(app) values for both HMG boxes B and B7 were ~25 nM, indicating that the attachment of the C terminus to the minimal B-domain resulted in a significant increase of the DNA binding affinity with no further enhancement upon addition of the 7-residue N-terminal extension (Table I). Our DNA ring closure experiments, however, provide evidence that both the N- and C-terminal flanking sequences of the B-domain are required for efficient DNA bending by the B-domain.
In order to find out whether all seven amino acids (residues 85-91, four of them lysines) of the B-domain N-terminal extension (residues 85-180) are necessary for DNA bending, we performed circularization assays using B-domain polypeptides containing the same 18-residue C-terminal extensions but having N-terminal extensions of different lengths. Addition of Asp to the extreme N terminus of the HMG box B (D-HMG box B; box B1, residues 91-180) did not result in any increase in DNA circle formation relative to the HMG box B (Fig. 5, third and fourth lanes from the left). As shown in Fig. 5, extension of the N terminus of the HMG box B by Lys-Asp dipeptide (KD-HMG box B; box B2, residues 90-180) resulted in very intense formation of DNA circles. Interestingly, the estimated Kd(app) values for both HMG boxes B and B2 were similar at ~25 nM. Formation of DNA circles was not significantly affected by further N-terminal addition of either phenylalanine (FKD-HMG box B; box B3, residues 89-180), or phenylalanine plus three consecutive lysines (KKKFKD-HMG box B; box B6, residues 84-180) or Thr-Lys-Lys-Lys-Phe pentapeptide (TKKKFKD-HMG box B; box B7, residues 85-180), Fig. 5. These results suggest that a single lysine residue (Lys90) in a short N-terminal sequence 90KD91 attached to the B-domain is sufficient for strong bending distortion of DNA, provided the B-domain contains the 18-residue C-terminal flanking sequence.DNA Bending by the HMG1 A-domain-- HMG1 protein consists of two evolutionarily conserved, folded HMG box domains, A and B. Although the B-domain of HMG1 has been shown by several research groups to bend DNA (10, 11, 21, 22, 26), some authors have reported that the A-domain of HMG1 is not able to do so (10, 21, 22). We have therefore reinvestigated the DNA bending potential of the isolated A-domain of HMG1. Both the HMG box A (residues 1-84; Fig. 6) and a shorter A-domain (HMG box A', residues 1-78; data not shown) could mediate DNA circularization to approximately the same extent. However, comparison of intensities of DNA circles revealed that the A-domain (HMG box A) seemed to be less efficient in DNA bending (see also "Discussion") than the B-domain (HMG box B7) (Fig. 6). DNA ring closure assays by HMG1 and its HMG box domains are summarized in Table I.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of the present paper was to use the ligase-mediated cyclization assay to illuminate the effect of different flanking sequences of HMG1 box A- and B-domains on DNA bending. The cyclization assay has been used previously to detect HMG1-induced DNA bending in solution (10, 11, 21, 26). Although most of the HMG1 A- and B-domain polypeptides studied contained at their N termini the Gly-Ser dipeptide contributed by the parental GST vector, we also used in some DNA circularization experiments recombinant B-domain polypeptides (residues 89-176 and 92-176), which had been expressed in E. coli under the control of the T7 promoter/T7 RNA polymerase system. These were unfused and therefore lacked the extra glycine and serine residues at their N termini. The latter DNA circularization experiments conclusively demonstrated that the extra N-terminal Gly-Ser dipeptide of the recombinant B-domains did not significantly influence the results of the DNA ring closure assay.2 The fact that the HMG1 protein (10) and its isolated HMG box domains (as revealed from binding experiments with supercoiled plasmids; see Ref. 29 and Footnote 2) do not change helical twist of DNA argues against the possibility that the observed differences in intensities of DNA circles (this paper) are attributable to differences in torsional misalignment due to twists in the DNA, as opposed to differences in DNA bending. However, definitive conclusions regarding the extent of DNA bending by HMG1 or its HMG boxes will be reached using cyclization kinetics (30-32).
Results presented here establish that both the A- and the B-domains of the HMG1 protein are able to distort DNA by bending, albeit with markedly different ease (in particular at HMG concentrations 0.2-1.5 µM), thus resolving the debate concerning the DNA bending potential of the two HMG1 box domains (Refs. 10, 11, 21, and 22; formation of DNA circles was, however, very little promoted by the isolated HMG1 A- or B-domains at concentrations <0.2 µM2, where both the HMG1 (A+B)-bidomain and HMG1 could efficiently bend DNA (Fig. 3); see below). The apparent low DNA circle formation by the A-domain could be due to several factors. First, the HMG box A- and B-domains exhibit only ~30-40% similarity in amino acid sequence. Second, although both HMG1 box domains have a very similar global fold (33-35), there are small differences between the orientations of helix I in A- and B-domains, particularly in the C terminus relative to the rest of the helix (34, 35). Another explanation may be that there is a different sequence context between the two domains within the native HMG1 protein, particularly the existence of different flanking sequences of the two HMG box domains. As shown in this paper, both N- and C-terminal B-domain flanking sequences are a prerequisite for efficient DNA bending. Whereas the N-terminal flanking sequence of the B-domain is common to both A- and B-domains as a part of the linker region between them, the difference in the DNA bending potential of the two HMG1 boxes might be due to the presence of the basic C-terminal linker region flanking the B- and C-domains, which is absent from the A-domain. This may also contribute to the approximately double Kd(app) (lower DNA binding affinity) for the A-domain compared with the B-domain (HMG box B7).
The structure of the mouse LEF-1 HMG box complexed with DNA reveals that helix III of the sequence-specific HMG box has a kink, which enables extensive contacts between the basic C-terminal extension and the DNA (36). Neither the A- or B-domains of HMG1 have kinks in helix III (33-35, 37), and, consistent with this, no strong interactions between helix III of the A-domain of HMG1 and DNA were detected by NMR (35). Nevertheless, it is likely that the basic C-terminal flanking sequences of the HMG boxes could contribute to the binding of HMG1 to DNA (20) by facilitating DNA distortion by bending (this paper and Ref. 21). The fact that the N-terminal extension markedly increased the ability of the B-domain of HMG1 to bend DNA only if the C-terminal flanking sequence was present (this paper) could be a consequence of better folding of the minor wing of the HMG box, which has both extensions. The proximity of the N- and C-terminal extensions, which is readily seen in the published HMG1 box structures (33-35, 37), might also suggest that mutual contacts between the two ends, either directly or indirectly as a result of DNA binding, are important for DNA bending distortion.
Although the DNA bending potential of HMG1 appears to be due predominantly 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 affected in the native HMG1 protein by its acidic C-terminal domain. In the absence of any data regarding the tertiary structure of the native HMG1 protein, we can only speculate that covalent attachment of the A- and B-domains results in a spatial orientation of the two HMG box domains that brings basic regions of the A/B- and B/C-domains and the N terminus of the A-domain into close contact. It is likely that the latter interactions are under the control of the acidic C terminus of HMG1, since lack of such control over DNA bending properties of HMG1 is readily seen upon removal of the acidic C-domain. Whereas the ability of native HMG1 protein to bend DNA was only slightly effected by increasing its concentration, the HMG1 (A+B)-bidomain exhibited significant concentration dependence in its ability to promote DNA circularization. It is possible that the observed inhibition of DNA bending at higher HMG1 (A+B)-to-DNA ratios may be a consequence of the formation of compact DNA-HMG complexes (previously visualized by electron microscopy; Refs. 6 and 7), which could prevent the alignment of the two DNA ends by causing bending in opposite directions and/or reducing the accessibility of the ligase. The modulatory effect of the HMG1 acidic C-domain on DNA bending is consistent with its reported effects on DNA binding and supercoiling (4-8).
The ability of HMG1 or its HMG1 box proteins to induce DNA flexure has led to the proposal that the HMG box could act in vivo as a general DNA bending or looping domain (reviewed in Ref. 1). However, HMG1 and its boxes were also capable of recognizing and binding tightly to altered DNA conformations, such as underwound and prebent structures, four-way junctions, and DNA modified with the anticancer drug cisplatin (2, 15-17). It has been suggested that binding of HMG1 box proteins to prebent or altered DNA structures, in preference to B-forms of DNA, could selectively favor DNA structural recognition, e.g. by reducing trapping of sequence-specific HMG box-containing transcription factors by bent or distorted DNA structures, and/or stabilization over induction of DNA bending (reviewed in Ref. 1). It is interesting that the Lys90 of the N-terminal extension of the HMG1 B-domain, which is required for efficient DNA bending (this paper), is identical to the residue that forms a specific cross-link to platinum(II) in cisplatin-modified DNA (38). These results may indicate that Lys90 within the A/B linker of HMG1 is important for the HMG box B-domain both to bend and recognize DNA with a preformed bend. Whether only Lys90 or other residues of the B- and A-domains of HMG1, such as those of the N-terminal strand, helix I, and helix II, all of which have previously been reported to make extensive contacts with DNA (35, 37, 39-42), are involved in DNA bending is to be resolved by site-directed mutagenesis.
The cellular role of the HMG1 or HMG2 proteins remains elusive. However, their proposed functions in chromatin organization, DNA replication, transcription, recombination, and repair may include a number of interactions enabling recognition and modulation of DNA structures as well as participation in direct contacts with other proteins such as histones, transcription factors, and enzymes (2, 43-47). Here, it is relevant to mention the enhanced formation of linear multimers of DNA in the ligation of short linear DNA fragments in the presence of HMG1 or its HMG boxes (this paper). This probably reflects an increased proximity (molecular "crowding") of the DNA termini, which permits their efficient ligation into linear multimers. Although the biological significance and specificity of the stimulation of ligase activity by HMG1 or its HMG box domains remains to be elucidated with eukaryotic ligases, this function would not be incompatible with the proposed role of HMG1 box domain as a DNA chaperone.
![]() |
ACKNOWLEDGEMENTS |
---|
I thank Boena Krönerová
and Anna Kolíbalová for helpful assistance with the
cloning of some of the plasmid constructs and FPLC purification of
proteins, respectively. I am also grateful to my colleagues and
anonymous reviewers for critical reading of the manuscript and many
helpful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant A5004604 from the Internal Grant Agency of the Academy of Sciences of the Czech Republic and Grant 204/95/1373 from the Grant Agency of the Czech Republic.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 420-5-41517183;
Fax: 420-5-41211293.
1 The abbreviations used are: HMG, high mobility group; PCR, polymerase chain reaction; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; bp, base pair(s).
2
M. tros, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|