(Received for publication, October 3, 1994; and in revised form, December 27, 1994)
From the
Mammalian high mobility group HMG-I/Y chromatin proteins bind to
the minor groove of AT-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
A
T-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 A
T-rich DNA using diethylpyrocarbonate
suggests that the protein is able to recognize, bind to, and alter the
conformation of non-B-form DNA.
The HMG-I/Y proteins are small, nonhistone, chromosomal proteins
of the ``high mobility group'' (HMG). ()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 A
T-rich DNA in
vitro(5, 6, 7) . The sequences of
HMG-I/Y responsible for the interaction with A
T-rich DNA have
been identified(8) , and results from two-dimensional
H-NMR studies support the predicted
netropsin/distamycin-like structure of the DNA binding
domains(9) . In addition, the A
T minor groove binding
ligands netropsin, distamycin, and Hoechst 33258 have been shown to
compete with HMG-I/Y for binding to A
T-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
AT-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 AT-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.
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
AT-rich pBLT plasmid and its parent vector, pUC18. We interpret
this to mean that HMG-I/Y, in addition to its ability to bind
A
T-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 A
T-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 AT-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.
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.
Figure 3:
Effect of HMG-I(E91) on pBLT. A, Topoisomerase I-mediated relaxation assay of pBLT in the
presence of carboxyl-terminal deleted HMG-I(
E91) 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 E91 for supercoiled DNA using an assay based on the
competition of binding of a fluorescent dye, Hoechst 33258, to
A
T-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
E91 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, A
T-rich DNA(8) . The
affinities of HMG-I and
E91 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
E91 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 A
T-rich DNA, while at the same time
dramatically changing the ability of the protein to induce both
positive and negative supercoils in plasmid DNAs.
Figure 4:
DEPC footprint of supercoiled
pBLT-HMG-I(E91) 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.
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 AT-rich DNA(8) . The core of the binding domain
sequence, PRGRP, has been suggested to have a structure similar to that
of the A
T DNA binding drug netropsin(8, 45) , a
suggestion that is supported by circular dichroism(46) ,
molecular modeling(8) , and two-dimensional
H 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. ()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
A
T-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 A
T containing dodecamers based on
two-dimensional
H 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
1
mM) in comparison with complexes formed between intact HMG-I
and supercoiled (K
25-35 nM, Table 1) or linear A
T-rich DNA (K
1 nM,(8) ). The consensus binding domain alone has
a K
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 E91 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 (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-
B, 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 AT-rich, supercoiled pBLT DNA was reacted
with DEPC in the absence or presence of HMG-I(
E91). 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 A
T-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 A
T-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 A
T-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 A
T-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.