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Volume 270, Number 9, Issue of March 3, 1995 pp. 4355-4360
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Changes in Superhelicity Are Introduced into Closed Circular DNA by Binding of High Mobility Group Protein I/Y (*)

(Received for publication, October 3, 1994; and in revised form, December 27, 1994)

Mark S. Nissen (1),  Raymond Reeves (1) (2)(§)

From the  (1)Department of Biochemistry and Biophysics and the (2)Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4660

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mammalian high mobility group HMG-I/Y chromatin proteins bind to the minor groove of AbulletT-rich DNA sequences with high affinity both in vivo and in vitro. Topoisomerase I-mediated relaxation assays, analyzed by one- and two-dimensional agarose gel electrophoresis, indicate that binding of recombinant human HMG-I/Y to closed circular DNA introduces positive supercoils at low protein to nucleotide molar ratios and negative supercoils at higher ratios. This is interpreted to mean that HMG-I/Y binding initially causes bending of the DNA helix followed by unwinding of the helix. In contrast, binding of another minor groove binding ligand, netropsin, introduces positive supercoils only. An in vitro produced mutant HMG-I/Y protein lacking the negatively charged carboxyl-terminal domain binds AbulletT-rich DNA approximately 1.4-fold better than the native protein, yet it is estimated to be 8-10-fold more effective at introducing negative supercoils. This finding suggests that the highly acidic C-terminal region of the HMG-I/Y protein may function as a regulatory domain influencing the amount of topological change induced in DNA substrates by binding of the protein. Footprinting of HMG-I/Y on negatively supercoiled AbulletT-rich DNA using diethylpyrocarbonate suggests that the protein is able to recognize, bind to, and alter the conformation of non-B-form DNA.

INTRODUCTION

The HMG-I/Y proteins are small, nonhistone, chromosomal proteins of the ``high mobility group'' (HMG). (^1)Members of this family include the isoform proteins HMG-I and HMG-Y (1, 2) and the homologous protein HMG-I(C)(3) . These proteins are distinguished from other HMG (4) proteins by their ability to recognize and specifically interact with the minor groove of AbulletT-rich DNA in vitro(5, 6, 7) . The sequences of HMG-I/Y responsible for the interaction with AbulletT-rich DNA have been identified(8) , and results from two-dimensional ^1H-NMR studies support the predicted netropsin/distamycin-like structure of the DNA binding domains(9) . In addition, the AbulletT minor groove binding ligands netropsin, distamycin, and Hoechst 33258 have been shown to compete with HMG-I/Y for binding to AbulletT-rich DNA, suggesting they posses a structure similar to the HMG-I/Y DNA binding domains (8, 10) and may bind DNA in a similar fashion.

In vivo, HMG-I/Y has been immunolocalized to the AbulletT-rich G/Q and C bands of mammalian metaphase chromosomes(11, 12) , suggesting a structural role for the protein, and good evidence has been presented linking HMG-I/Y with activation of chromatin domains via displacement of histone H1 from scaffold attachment regions(13, 61) . In addition to possible roles as a chromatin structural factor, recent reports indicate that HMG-I/Y also functions as a general transcription factor (14, 15, 16, 17, 18) . With respect to these observations, elevated in vivo levels of HMG-I/Y have been correlated with both neoplastic transformation(1, 2, 3, 20, 21, 22, 23) and with metastatic tumor progression (24, 25, 26) . Furthermore, proteins otherwise unrelated to HMG-I/Y have been discovered that contain amino acid sequences with similarity to the DNA-binding domains. Examples include HRX (ALL) in humans(27) , D1 in Drosophila(62) , ATBP-1 from pea(28) , LAT1, NAT1, and 2 from soybean(29) , PF1 from rice(30) , and MIF2 (31) and datin (63) from Saccharomyces cerevisiae.

In this report, we extend our earlier studies of HMG-I/Y binding to naked AbulletT-rich DNA and to chicken mononucleosomes to include the effect of protein binding on the topological state of closed circular DNA. Topoisomerase I-mediated relaxation assays of complexes of HMG-I/Y with supercoiled plasmid DNA reveal that HMG-I/Y induces positive supercoiling at low protein to nucleotide molar ratios in a manner similar to netropsin, suggesting that bends are induced in the substrate at these protein concentrations. At higher ratios, however, negative supercoils are induced, indicating that protein binding leads to underwinding of DNA at these concentrations, in contrast to the positive supercoils induced by binding of netropsin. Additionally, we present footprinting evidence that HMG-I/Y is capable of binding to and altering the secondary structure of non-B-form DNA.

EXPERIMENTAL PROCEDURES

Preparation of HMG-I(Y) Proteins and DNA

Recombinant human HMG-I and HMG-Y were produced using the expression vector pET7C and were purified by ion exchange chromatography as described previously (32) . Protein concentrations were determined spectrophotometrically using = 74,000 and = 68,000 liters/molbulletcm for HMG-I and HMG-Y, respectively(8) . An additional HMG-I protein, DeltaE91, was also prepared using the same methods. This protein lacks the 17 glutamic acid-rich residues at the carboxyl terminus of the intact HMG-I molecule, and its construction has been detailed previously(33) . The construction of plasmid pBLT, which contains the 300-base pair AbulletT-rich 3`-untranslated region derived from the bovine interleukin-2 cDNA, has been described(8) . Plasmids pUC-18 and pBLT were purified by banding on CsCl gradients, and concentrations were determined by absorption at 260 nm.

Preparation of Topoisomerase I

Washed, packed, and frozen chicken erythrocytes were purchased as 10-ml aliquots from Lampire Biological, Pipersville, PA. Cells were simultaneously thawed and lysed in ice-cold 10 mM MOPS, pH 7.2, 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride and 0.2% Nonidet P-40. Nuclei were pelleted by centrifugation at 2000 times g for 10 min. The supernatant was carefully removed, and the nuclear pellet was washed by resuspension in buffer without Nonidet P-40 and subsequent centrifugation. Topoisomerase I was then isolated from washed nuclei, employing the procedure detailed by Pfaffle and Jackson (34) . Fractions containing topoisomerase activity were pooled and split into aliquots. These aliquots were stored frozen at -70 °C. Enzyme activity was assayed using pUC18, where one unit will relax 0.5 µg of supercoiled DNA in 0.5 h at 37 °C.

Topoisomerase I-mediated Relaxation Assay

For a typical assay, either HMG-I or HMG-Y protein, ethidium bromide (EtBr) or netropsin was added in varying ratios to 2 µg of negatively supercoiled pBLT or pUC18 plasmid, and incubated at 23 °C for 30 min in 200 µl of 20 mM TrisbulletCl, pH 8, 50 mM NaCl, 50 µg/ml bovine serum albumin, 5% glycerol, and 1 mM dithiothreitol. Twenty units of topoisomerase I were added, and the samples were incubated for 1 h at 37 °C. Samples were then made 0.1% in SDS and digested with 0.1 mg/ml Proteinase K for 30 min at 37 °C followed by extensive extraction with phenol/CHCl(3) and ethanol precipitation of the DNA. The DNA was redissolved in TE (10 mM Tris, pH 7.6, 10 mM EDTA) and electrophoresed overnight on 1.5% agarose-TAE gels at 0.5 V/cm and 23 °C. The gels were stained with EtBr and photographed.

Two-dimensional Chloroquine Gels

Aliquots of topoisomerase-relaxed DNA prepared for one-dimensional analysis were loaded onto 1.5% agarose-TAE (40 mM Tris, pH 8.3, 20 mM sodium acetate, 2 mM EDTA) gels and electrophoresed for 3 h at 6 V/cm and 23 °C. The gels were then stained for 1 h in TAE containing 1.5 µg/ml chloroquine phosphate, rotated 90° with respect to the original direction of migration, and the electrophoresis was repeated in TAE supplemented with 1.5 µg/ml chloroquine phosphate. The gels were stained with EtBr and photographed.

Determination of Protein-DNA Dissociation Constants

Dissociation constants of HMG-I and HMG-I(DeltaE91) to supercoiled plasmids were determined using a fluorescence competition assay employing the dye Hoechst 33258 as reported previously(8, 35) . For these assays, plasmid concentration was fixed at 0.33 µg/ml, protein concentration was 10 nM, and dye concentrations ranged from 0 to 50 nM.

Footprints of Supercoiled DNA

Footprinting of supercoiled DNA with diethyl pyrocarbonate (DEPC) was largely as described by McLean(36) . Various amounts of HMG-I(DeltaE91) were mixed with 1 µg of supercoiled pBLT in 45 µl of TE. Following a 30-min incubation at 23 °C, 5 µl of DEPC was added, and incubation continued for an additional 15 min. The samples were then precipitated with sodium acetate and ethanol and redissolved in water. After addition of the appropriate buffer, the DNA was digested with EcoRI, and the ends were labeled by standard methods (37) using [alpha-P]dATP and Klenow polymerase. The labeled 300-base pair fragment was then released from the vector by digestion with SalI, isolated by agarose gel electrophoresis, and purified from the agarose with Geneclean (Bio 101, La Jolla, CA). The purified DNA was finally cleaved by treatment with 10% piperidine for 15 min at 90 °C and the cleavage products were visualized by autoradiography following electrophoresis on a 6% sequencing gel. Autoradiographs were scanned with an LKB Ultroscan XL laser densitometer.

RESULTS

Topoisomerase-mediated Relaxation Assays

The effect of various DNA binding ligands on the superhelicity of two plasmid substrates was investigated by agarose gel electrophoresis. In these experiments, ligand-DNA complexes were treated with topoisomerase I to relax the superhelical stress in the DNA followed by removal of the ligand and subsequent electrophoretic resolution of the resulting topoisomers. Topoisomerase I removes any superhelical stress that is not constrained by ligand-DNA interactions. Consequently, the topoisomers resolved following removal of the ligand reflect changes in DNA secondary and tertiary structure due to interaction with the ligand. Plasmid pBLT was chosen as a test DNA substrate because it contains a 300-base pair AbulletT-rich insert whose interaction with HMG-I/Y has been previously characterized(8, 38) . The AbulletT-rich insert was derived from the 3`-untranslated region of the bovine interleukin-2 cDNA and has an AbulletT base content of 71.4 mol % discounting the poly(A) tail. Plasmid pUC18 was used for sake of comparison since pBLT was derived from it.

Fig. 1shows the one-dimensional electrophoretic results typical for a topoisomerase assay. Lane1 of each panel shows the plasmid relaxed with topoisomerase I in the absence of protein. The relative positions of relaxed and supercoiled plasmid are indicated in the figure. In panelA, the two plasmids were relaxed with topoisomerase in the presence of molar ratios of HMG-I to nucleotide that varied from 0 to 2.2. It should be noted that in these assays, the plasmids are initially supercoiled when HMG-I/Y is bound and that topoisomerase is added subsequently. Consequently, non-B-form DNA conformations, which might be formed as a result of supercoiling, could also serve as recognition sights for protein binding. It can be clearly seen that the presence of increasing amounts of HMG-I protein results in a change in the distribution of the topoisomers formed following relaxation of either plasmid and subsequent removal of the protein. PanelB of the figure shows results of an assay performed with HMG-Y. Comparison of panelsA and B indicates that increasing concentrations of both HMG-I and HMG-Y produce identical distributions of topoisomers in either pBLT or pUC18. Interestingly, HMG-I and HMG-Y produced qualitatively similar distributions of topoisomers in both the AbulletT-rich pBLT plasmid and its parent vector, pUC18. We interpret this to mean that HMG-I/Y, in addition to its ability to bind AbulletT-rich sequences and influence the secondary and tertiary structure of the plasmid, may also recognize and bind structures induced in substrate DNA as a result of supercoiling. It should also be noted that the distribution of isomers over the course of the titration is different for the two plasmids, suggesting that the AbulletT-rich insert of supercoiled pBLT may have some intrinsic structure or that HMG-I/Y interacts differently with it.

Figure 1: Topoisomerase-mediated relaxation assays. Plasmids pBLT and pUC18 were relaxed with topoisomerase I in the presence of different DNA binding ligands. In each of the four panels, the leftset of lanes represents pBLT, and the rightset represents pUC18. In all cases, ligand concentration increases from left to right. The relative positions of form I and II DNA are indicated. A, HMG-I at protein to nucleotide molar ratios of 0, 0.27, 0.54, 0.81, 1.1, 1.6, and 2.2; B, HMG-Y at the same protein to nucleotide ratios as in A; C, EtBr at 0, 0.05, 0.075, 0.1, 0.25, 0.5, and 1 µg/ml; D, netropsin at 0, 0.0075, 0.01, 0.025, 0.05, 0.1, and 0.2 molar ratio of drug to nucleotide.


The same experiment was carried out using the AbulletT-binding antibiotic netropsin as the DNA binding ligand. Netropsin was chosen for two reasons. First, it has been suggested that the DNA binding domain of HMG-I may mimic the structure of netropsin (8, 39) and that the two molecules may interact with DNA in a similar fashion. Second, it has also been demonstrated that netropsin introduces positive supercoils into DNA when subjected to the sort of assay employed here(40, 41) . As shown in Fig. 1C, relaxation of the plasmid in the presence of netropsin followed by removal of the antibiotic results in changes in the superhelical density of the DNA that are qualitatively similar to those produced by HMG-I/Y. In order to help determine the sign of the supercoils introduced by HMG-I/Y, topological ``standards'' were prepared by topoisomerase-mediated relaxation of plasmid DNA in the presence of the intercalating dye EtBr. The binding of EtBr to DNA has been extensively characterized, and it is well known that intercalation of EtBr causes unwinding of the helix(42, 43) . It is also known that relaxation of circular DNA in the presence of EtBr followed by removal of the dye results in negatively supercoiled DNA; an effect opposite of that produced by netropsin(40) . Fig. 1D demonstrates the results of relaxation of pBLT and pUC18 in the presence of increasing concentrations of EtBr.

Determination of the Sign of Supercoiling

Aliquots of the samples shown in Fig. 1were electrophoresed on two-dimensional agarose gels in the presence of 1.5 µg/ml of chloroquine in the second dimension. Chloroquine intercalates into DNA and, under the conditions employed here, will retard the migration of negatively supercoiled DNA while it increases the migration of relaxed and positively supercoiled DNA(44) . This effect is seen in Fig. 2. In the figure, negatively supercoiled topoisomers tend to the left of the figure (the origin of the second dimension), while positively supercoiled topoisomers tend to the right (the direction of migration in the second dimension). Fig. 2, A and B, shows two-dimensional gels of pBLT and pUC18 relaxed in the presence of HMG-I and HMG-Y, respectively. In both cases, it is seen that with increasing protein concentration, the plasmids initially become positively supercoiled, that is, the migration rate of the topoisomers increases. However, as the protein concentration increases, the plasmids become negatively supercoiled as demonstrated by the reduced mobility of the ensemble of topoisomers. In contrast, pBLT relaxed in the presence of netropsin shows the opposite behavior (Fig. 2C). In this case, migration of the topoisomers is increased, indicating that the DNA is positively supercoiled; an observation in agreement with that reported previously(40, 41) . It is interesting to note that pUC18 exhibits both positive and negative supercoils at a netropsin concentration that produces all positive supercoils in pBLT, again suggesting that the ligand interacts differently with the AbulletT-rich BLT DNA. Fig. 2D shows the effect of EtBr on the distribution of topoisomers. It is evident that EtBr induces formation of negative supercoils in this assay. Overall, the distribution of topoisomers produced at low protein to nucleotide molar ratios resembles that produced by netropsin, while at higher protein to nucleotide molar ratios, the distribution resembles that produced by ethidium.

Figure 2: Two-dimensional chloroquine gels. Aliquots of samples shown in Fig. 1were run on 1.5% agarose gels, the gels were treated with 1.5 µg/ml chloroquine and run in a second dimension. The migration direction of each dimension is indicated. In each of the four panels, the upperset of bands represents pBLT and the lowerset of bands represents pUC18. Ligand concentration increases from left to right. The relative positions of form I and II DNA are indicated. A, HMG-I at protein to nucleotide molar ratios of 0, 0.27, 1.1, 1.6, and 2.2; B, HMG-Y at protein to nucleotide molar ratios of 0, 0.27, 0.81, 1.1, and 1.6; C, netropsin at 0, 0.025, 0.05, 0.1, and 0.2 drug to nucleotide molar ratio; D, EtBr at 0, 0.075, 0.1, 0.25, and 0.5 µg/ml.


Effect of Removal of HMG-I Carboxyl Terminus

Topoisomerase-mediated relaxation assays were performed using DeltaE91, an HMG-I protein that lacks the 17-glutamic acid-rich C-terminal residues found in the full-length protein(33) . Results of a typical one-dimensional assay are shown in Fig. 3A. The overall pattern of topoisomers produced by relaxation of DNA in the presence of the truncated protein is similar to that produced by the full-length protein (Fig. 1A). It is important to note, however, that the DeltaE91 to nucleotide molar ratios in Fig. 3are approximately 10-fold less than the HMG-I to nucleotide ratios in Fig. 1. The sign of supercoiling induced by DeltaE91 was determined by analysis on two-dimensional chloroquine gels (Fig. 3B), and the sign of the induced supercoils was determined to be positive at relatively low protein to nucleotide molar ratios, with negative supercoils being induced at higher protein to nucleotide molar ratios.

Figure 3: Effect of HMG-I(DeltaE91) on pBLT. A, Topoisomerase I-mediated relaxation assay of pBLT in the presence of carboxyl-terminal deleted HMG-I(DeltaE91) at protein to nucleotide molar ratios of 0, 0.04, 0.07, 0.11, 0.14, 0.20, and 0.27; B, two-dimensional gels containing 1.5 µg/ml of chloroquine in the second dimension. Protein to nucleotide molar ratios are 0, 0.07, 0.11, 0.22, and 0.27.


We investigated the relative affinity of both HMG-I and DeltaE91 for supercoiled DNA using an assay based on the competition of binding of a fluorescent dye, Hoechst 33258, to AbulletT-rich HMG-I binding sites(8) . For this determination, supercoiled plasmid DNA at a fixed concentration was titrated with Hoechst 33258 in the absence or in the presence, of 10 nM HMG-I or DeltaE91 in buffer containing 50 mM NaCl. In Table 1, it is seen that the affinity of Hoechst 33258 for supercoiled DNA is between 13 and 18 nM, in good agreement with a dissociation constant of 9.6 nM previously reported for binding of the dye to linear, AbulletT-rich DNA(8) . The affinities of HMG-I and DeltaE91 for supercoiled pUC18 were very similar with values of 35.5 nM and 32.4 nM, respectively. In contrast, the affinity of HMG-I for supercoiled pBLT was determined to be 38.0 nM, and that of DeltaE91 was determined to be 26.6 nM, a change in affinity of about 1.4-fold. This suggests that the acidic carboxyl-terminal of HMG-I is capable of modulating, to a relatively small degree, the binding of the protein with supercoiled AbulletT-rich DNA, while at the same time dramatically changing the ability of the protein to induce both positive and negative supercoils in plasmid DNAs.

DEPC Footprints of Supercoiled DNA

The AbulletT-rich insert of supercoiled pBLT was footprinted with DEPC either as naked DNA or as a complex with DeltaE91. Fig. 4compares the relative cleavage frequency at adenine of supercoiled DNA alone or of supercoiled DNA complexed with DeltaE91 (this experiment was not performed on pUC18). It is apparent that certain regions of the AbulletT-rich pBLT DNA react readily with DEPC, suggesting that the DNA is not in the refractory ``normal'' B conformation but possesses an altered secondary structure, which renders it more susceptible to reaction with the reagent(36) . It can also be seen that addition of DeltaE91 protein causes a dramatic change in the cleavage pattern of the DNA and that this change involves the regions most susceptible to modification by DEPC (Fig. 4, bracketedpeaks). In contrast, AbulletT-rich regions of the BLT DNA that have been previously identified by footprinting to be HMG-I binding sites(8, 38) show little or no change in their DEPC footprint (Fig. 4). The data suggest that, in addition to the characterized ability to bind to the minor groove of AbulletT-rich DNA, HMG-I/Y proteins may also recognize and bind non-B-form DNA and that such binding may, in turn, further alter the conformation of the bound DNA.

Figure 4: DEPC footprint of supercoiled pBLT-HMG-I(DeltaE91) complex. Supercoiled pBLT was reacted with diethyl pyrocarbonate in the absence or the presence of carboxyl-terminal deleted HMG-I at a protein to nucleotide molar ratio of 0.27. Following modification, the protein was removed, the DNA was digested with EcoRI and end-labeled with P. The labeled fragment was subsequently isolated and cleaved with piperidine, and the resulting fragments were separated by electrophoresis. The figure shows laser densitometer scans of an autoradiograph.


DISCUSSION

Earlier work from this laboratory has identified an 11-amino acid peptide, with the consensus sequence TPKRPRGRPKK, as the sequence responsible for the specific interaction of HMG-I/Y with the minor groove of AbulletT-rich DNA(8) . The core of the binding domain sequence, PRGRP, has been suggested to have a structure similar to that of the AbulletT DNA binding drug netropsin(8, 45) , a suggestion that is supported by circular dichroism(46) , molecular modeling(8) , and two-dimensional ^1H NMR studies(9) . This notion is further strengthened by the fact that netropsin and the structurally similar drug distamycin bind to DNA sequences that are preferred HMG-I/Y binding sites and will, in fact, compete with the protein for these sites(10, 47) . Prior studies on the binding of netropsin to negatively supercoiled DNA established that relaxation of the drug-DNA complex by topoisomerase I followed by removal of the drug resulted in the introduction of positive supercoils(40) . A more recent study compared the effects of minor groove binding drugs including netropsin, distamycin, pentamidine, and others. The investigators concluded that different minor groove binding ligands differ in their ability to induce changes in supercoiling and that these differences are not due to a single binding property of each ligand(48) . The present study was initiated to examine the effect of binding of HMG-I/Y on DNA supercoiling and to compare it to the known effect of netropsin binding.

The fundamental relationship between the topological and geometrical properties of closed circular DNA is described by the expression Lk = Tw + Wr where Lk is the topological linking number, Tw is the twist and Wr is the writhe(49) . In the case of netropsin, Storl et al.(48) suggest that netropsin binding causes bending of the helix axis leading to changes in Tw and Wr and, following relaxation by topoisomerase, a change in Lk. The appearance of positive supercoils, an increase in Lk, is attributed to an increase in Tw. In support of this explanation, x-ray crystallographic analysis of netropsin-DNA complexes shows that netropsin binding causes an approximate 8° bend in the helix axis and a widening of the minor groove(50, 51) . The results presented in Fig. 1and Fig. 2indicate that HMG-I/Y introduces positive supercoils into closed circular DNA as measured by these assays. Indeed, the effect of HMG-I/Y on the superhelicity of the plasmids is, at low protein to nucleotide molar ratios, qualitatively similar to the effect of bound netropsin, suggesting that HMG-I/Y binding to high affinity sites results primarily in bending of the helix axis. This finding is consistent with our observations that in certain circularly permuted plasmid vectors, HMG-I/Y appears to induce DNA bending. (^2)At higher protein to nucleotide molar ratios, the effect is more like that of EtBr. In the case of EtBr, binding of the ligand results in untwisting of the DNA (i.e.Tw decreases) accompanied by an equal increase in Wr since Lk must remain constant. Following relaxation of the complex with topoisomerase I, Wr = 0 and, from the above relationship, Lk = Tw. Removal of the intercalator results in increased Tw and the consequent generation of negative Wr, which is seen as negative supercoils(49) . In contrast to netropsin, the data suggests that at relatively high ligand to nucleotide ratios, HMG-I/Y binding causes unwinding of the helix in addition to bending. This effect due to helix unwinding is probably a cumulative phenomena, explaining why it is only seen at higher protein to nucleotide ratios. This suggestion is in agreement with an earlier circular dichroism study that found that HMG-I binding significantly altered the conformation of AbulletT-rich DNA(46) . In marked contrast, however, is a recent report of the interaction of the HMG-I/Y binding domain core sequence with synthetic AbulletT containing dodecamers based on two-dimensional ^1H NMR measurements(52) . According to this study, the peptide sequence RGR mimics netropsin structurally, and binding of the core sequence does not perturb the DNA conformation. It should be noted that the complex formed by the test peptide, PRGRP, and DNA used in that study is very weak (K(d) approx 1 mM) in comparison with complexes formed between intact HMG-I and supercoiled (K(d) approx 25-35 nM, Table 1) or linear AbulletT-rich DNA (K(d) approx approx1 nM,(8) ). The consensus binding domain alone has a K(d) of about 10 µM(8) . The 3-6 orders of magnitude difference in dissociation constants indicates that more than just the core binding domain is involved in tight complex formation and suggests that other modes of binding may be operational in the intact protein that are responsible for the observed DNA conformational changes.

When we investigated the ability of the C-terminal deleted DeltaE91 protein to induce DNA supercoiling, we were surprised to find that the truncated protein was about 10-fold more active than intact HMG-I/Y (Fig. 3). A very similar observation has recently been made with the unrelated high mobility group proteins HMG-1 and HMG-2(53, 54) . These proteins have highly acidic C-terminal domains: a continuous run of 30 aspartate or glutamate residues in the case of HMG-1(55) . Proteolytic removal of the C-terminal domain of the HMG-1 and -2 proteins has been shown to increase the affinity of the protein for DNA between 2- (53) and 4-fold (54) and to consequently increase the ability of the protein to induce negative supercoils in closed, circular DNA. The increase in affinity and supercoiling ability has been attributed to ``electrostatic modulation'' of the positively charged DNA binding domains by the negatively charged acid tail(53, 54) . In the case of HMG-I/Y, we observe a smaller increase in affinity of about 1.4-fold upon removal of 8 noncontinuous glutamate residues located in the C-terminal 17 amino acid residues of human HMG-I, concomitant with an estimated 8-10-fold increase in the positive and negative supercoiling ability. Why the increase in supercoiling activity is not more closely correlated with the increase in affinity, as in the case of HMGs 1 and 2, is currently unknown. Nevertheless, these results suggest that the negatively charged C-terminal domain of HMG-I/Y may function as a regulatory element that influences the topological state of DNA in protein-DNA complexes.

It has recently been established that HMG-I/Y functions as an in vivo transcriptional factor (14, 15) and is required for induction of the human interferon beta (15) and IL-2R genes(56) . HMG-I/Y has been postulated to induce bending of the DNA axis and has been demonstrated to participate in protein-protein interactions with transcription factors NF-kappaB, ATF-2(15, 16) , and Elf-1(56) . In light of the information presented here, we suggest that, aside from any potential DNA bending by the protein(15, 16, 56) , HMG-I may also cause helix unwinding or some similar change in DNA secondary structure which, in some cases, facilitates assembly of other factors into a functional transcriptional complex. Furthermore, it is tempting to speculate that the demonstrated interaction of HMG-I/Y with the basic zipper region of ATF-2 (16) may be mediated by the acid tail of HMG-I/Y. If this interaction neutralizes the charge of the acid tail, the alteration of DNA secondary structure by HMG-I/Y may be potentiated.

Results of the footprinting study suggest that HMG-I/Y may posses previously unrecognized DNA recognition and binding activities. Diethyl pyrocarbonate was chosen as a footprinting reagent because it does not produce a ``traditional'' footprint, i.e. a footprint based on protection of DNA by ligand. DEPC reacts with N-7 of adenine to form a base-labile adduct and allows modification, without cleavage, of DNA in a DNA-protein complex. N-7 of adenine, which projects into the major groove of B-form DNA, is rendered insensitive to DEPC because of stacking interactions between adjacent bases(36) . Consequently, if these stacking interactions are perturbed by a change in DNA conformation (e.g. single-stranded, bent, or overwound DNA), N-7 may become more accessible to DEPC, and the increased reactivity can be used to map regions of altered DNA structure(36) . This can be seen in Fig. 4, in which AbulletT-rich, supercoiled pBLT DNA was reacted with DEPC in the absence or presence of HMG-I(DeltaE91). In the absence of protein, it is apparent that there are regions of the pBLT DNA that show enhanced reactivity with DEPC. Indeed, there is what appears to be a DEPC hypersensitive region near the 5` end of the DNA (shown under the bracket in Fig. 4), and this region may correspond to non-B-form DNA that is formed by the plasmid to relieve the unfavorable free energy associated with negative supercoiling(49) . It is striking to see that HMG-I/Y, while known to bind to numerous AbulletT-rich regions in this DNA(7, 8) , seems to preferentially recognize and alter the conformation of the hypersensitive region as detected by DEPC reactivity. Other examples of this behavior involving the binding of small, HMG-I/Y-like molecules to DNA are known. It has been shown that high concentrations of netropsin and the AbulletT-DNA binding drug distamycin are capable of driving the A to B-form transition, presumably by stabilization of the B-form (58) and that increasing amounts of bound distamycin cause conformational changes in DNA(59) . A recent investigation of distamycin binding to 5 S ribosomal RNA genes of Xenopus indicates that the drug first occupies an AbulletT-rich site that is interrupted by a G/C base pair, suggesting that the drug is recognizing altered DNA structure (60) . In addition, with increasing distamycin concentration, the position of the binding sites were observed to change, indicating that the drug was affecting the DNA structure(60) . HMG-I/Ys ability to recognize non-B-form DNA structure in addition to the minor groove of AbulletT-rich sequences, and its ability to differentially alter DNA conformation, may be integral to the protein's activity as a transcriptional control factor and as a chromatin structural element.

FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant 5-R01-AI26356 and National Science Foundation Grant DCB-8904408 (both to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 509-335-1948; Fax: 509-335-9688.

(^1)
The abbreviations used are: HMG, high mobility group nonhistone chromatin protein; MOPS, 4-morpholinepropanesulfonic acid; DEPC, diethyl pyrocarbonate.

(^2)
M. S. Nissen and R. Reeves, unpublished observations.

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