From the Departments of
Biochemistry/Biophysics and
§ Genetics and Cell Biology, Washington State University,
Pullman, Washington 99164-4660
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ABSTRACT |
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Chromosomal translocations involving genes coding
for members of the HMG-I(Y) family of "high mobility group"
non-histone chromatin proteins (HMG-I, HMG-Y, and HMG-IC) have been
observed in numerous types of human tumors. Many of these gene
rearrangements result in the creation of chimeric proteins in which the
DNA-binding domains of the HMG-I(Y) proteins, the so-called A·T-hook
motifs, have been fused to heterologous peptide sequences. Although
little is known about either the structure or biophysical properties of
these naturally occurring fusion proteins, the suggestion has been made
that such chimeras have probably assumed an altered in vivo
DNA-binding specificity due to the presence of the A·T-hook motifs.
To investigate this possibility, we performed in vitro "domain-swap" experiments using a model protein fusion system in
which a single A·T-hook peptide was exchanged for a corresponding length peptide in the well characterized "B-box" DNA-binding domain of the HMG-1 non-histone chromatin protein. Here we report that chimeric A·T-hook/B-box hybrids exhibit in vitro
DNA-binding characteristics resembling those of wild type HMG-I(Y)
protein, rather than the HMG-1 protein. These results strongly suggest
that the chimeric fusion proteins produced in human tumors as a result
of HMG-I(Y) gene chromosomal translocations also retain
A·T-hook-imparted DNA-binding properties in vivo.
The mammalian HMG-I(Y) family of "high mobility group"
(HMG)1 chromatin proteins are
founding members of a new assemblage of nuclear proteins referred to as
"architectural transcription factors," whose role in gene
regulation is the recognition and modulation of both DNA and chromatin
structure (recently reviewed in Ref. 1). The human HMG-I(Y) protein
family consists of: HMG-I (11.7 kDa), HMG-Y (10.6 kDa), and HMGI-C (12 kDa). HMG-I and HMG-Y are isoform proteins (identical in sequence
except for an internal 11-amino acid deletion in the latter) derived
from an alternatively spliced mRNA transcript coded for by the
HMG-I(Y) gene at chromosomal locus 6p21 (1-5). The related
HMGI-C protein is coded for by a separate gene located at chromosomal
locus 12q14-15 (6). Members of the HMG-I(Y) family share many
biochemical and biophysical properties, including possessing three
independent "A·T-hook" DNA-binding motifs, which exhibit a marked
preference toward A·T-rich B-form DNA (2, 3, 7-9). Therefore, for
convenience, we will collectively refer to members of this family as
simply HMG-I(Y), unless otherwise noted. Various lines of evidence
indicate that the individual A·T-hook motifs of the HMG-I(Y) proteins
recognize the structure associated with the narrow minor groove of
A·T-rich target DNA rather than its nucleotide sequence (1, 8, 10, 11). The physical basis for this DNA-structural recognition became
clear with NMR structural studies, which indicated that in solution the
HMG-I(Y) protein is unstructured but that upon substrate association
the A·T-hook undergoes a disorder-to-order structural transition
necessary for specific A·T-DNA binding (9, 12). During this
transition, the arginine side chains of the palindromic "core"
sequence, Pro-Arg-Gly-Arg-Pro (PRGRP; Fig. 1) assume a specific planar,
crescent-shaped structure that confers selectivity for the narrow minor
groove of A·T-rich sequences. This core peptide motif is the most
highly conserved peptide sequence found in the HMG-I(Y) family of
proteins. Correspondingly, the A·T-hook motif is evolutionarily
conserved as a DNA-binding unit in many other proteins and
transcription factors found in organisms ranging from bacteria (13) to
humans (1).
Chromosomal translocations of the genes coding for the HMG-I(Y)
proteins are among the most common rearrangements observed in human
neoplasms (reviewed in Ref. 14). In the majority of cases, these
chromosomal translocations result in the creation of chimeric genes
coding for hybrid proteins in which the A·T-hook motifs derived from
one of the HMG-I(Y) gene loci are fused "in frame" to the
N-terminal end of ectopic peptide sequences derived from a number of
other gene loci. Such rearrangements are quite prevalent in benign
mesenchymal human tumors including lipomas, leiomyomas, fibroadenoma,
pleomorphic adenomas, aggressive angiomyxomas, and pulmonary hamartomas
(14). Table I lists some examples of aberrant chimeric proteins
containing HMG-I(Y)-like A·T-hook motifs. However, little information
is available about these fusion protein's role, if any, in the process
of tumorigenesis. Nevertheless, it has been suggested that one possible
in vivo role for these ectopic fusion sequences in tumor
cells is to interfere with the normal gene regulatory function of the
HMG-I(Y) protein (15). Another suggestion is that a likely consequence
of the attachment of the A·T-hook motifs to other proteins or
peptides is to change the DNA-binding characteristics of the hybrids so
that they now "mistarget" to new substrate binding sites within the
nucleus (16-18). In either case, the intrinsic assumption is that the
A·T-hook peptides present in chimeric proteins retain their normal
A·T-DNA substrate binding specificity. In lieu of direct experimental support in favor of such an assumption, it is just as reasonable, however, to suspect that the substrate binding properties of the A·T-hook motif are significantly altered when fused to a large, structured ectopic protein partner.
Unfortunately, it is difficult to assess whether or not the A·T-hook
DNA-binding motifs found in naturally occurring chimeric tumor proteins
are functional since little is known about either the biological
function or structure of these fusion hybrids. To directly test the
functionality of the A·T-hook motif in the context of a structured
fusion partner, we performed in vitro domain swap
experiments (see Fig. 1) in which the second, and strongest
DNA-binding, A·T-hook motif of the HMG-I(Y) protein (9) was inserted
in place of a corresponding length peptide at the N terminus of the
HMG-1 B-box DNA binding domain (1). The HMG-1 B box peptide was chosen
as a "model" fusion partner for our domain swap experiments with
HMG-I(Y) because NMR studies have demonstrated that the native wild
type B-box peptide is highly structured in solution (Fig. 1) (19, 20),
and because an extensive literature also exists on the possible
biological function(s) of proteins that contain one or more HMG-1 box
DNA-binding motifs.
Here we report on the biophysical and substrate binding properties of
recombinant chimeric HMG-I(Y) A·T-hook/HMG-1 B-box
proteins. Proper folding of both the hybrid B-box (hereafter referred
to as the "correctly folded hybrid") and the wild type (WT) B-box
domains were verified using circular dichroism and intrinsic
fluorescence measurements. As a control, a non-folded hybrid
B-box protein, also containing a single A·T-hook, was likewise generated and analyzed in comparison to both the correctly folded hybrid and the WT B-box proteins using electrophoretic mobility shift
assays. These experiments demonstrate that both the correctly folded
and the non-folded hybrid B-box proteins have acquired DNA-binding
specificities more closely resembling those of the HMG-I(Y) proteins
than those of either the full-length HMG-1 protein or those of the
isolated WT HMG-1 B-box domain. The ability to alter the substrate
binding preferences of the WT HMG-1 B-box protein by motif exchange
with an A·T-hook DNA-binding peptide suggests that the chimeric
fusion proteins observed in vivo in human tumors also retain
HMG-I(Y)-like substrate binding specificities. In addition, the results
presented provide new insight into the nature of interactions of both
the HMG-1 B-box and HMG-I(Y) A·T-hook with DNA.
5' Construction of Glutathione S-Transferase (GST) Fusion Protein
Expression Vectors for Producing Wild Type and Correctly Folded Hybrid
HMG-1 B-box Recombinant Proteins--
The isolated WT HMG-1 B-box
domain, corresponding to residues 89-164 of the 214-amino acid
full-length HMG-1 protein, was constructed by PCR amplification of a
human HMG-1 cDNA template (21) using primers 1 and 2 listed above.
The correctly folded hybrid B-box was constructed in three consecutive
steps. 1) The DNA region coding for the HMG-1 B-box was PCR-generated
lacking the 10-amino acid (aa) N-terminal extended segment (residues
89-98) with primers 3 and 2, hereafter referred to as the "B-box
w/o N-term" fragment; 2) PCR primers 4 and 5 were used to
amplify the region of the human HMG-I(7C) cDNA (5) coding for the
"second" A·T-hook DNA-binding motif (i.e. PTPKRPRGRP)
corresponding to aa residues 52-61 of full-length HMG-I; 3) the
correctly folded hybrid B-box construct was then created by annealing
the isolated PCR DNA fragments coding for the A·T-hook motif and the
B-box w/o N-term and extending the annealed products by PCR
using primers 4 and 2. The 5' region of the B-box w/o N-term corresponding to primer 3 contained sequence complimentary to the 3'
portion of A·T-hook motif, and the 3' region of primer 5 contained
sequence complimentary to the 5' portion of the B-box w/o
N-term construct. Specific nucleotide base mutations in the third base
wobble position of the original HMG-I(7C) and HMG-1 cDNA clones
that that do not alter amino acid coding sequence were engineered into
the PCR primers to counteract primer-dimer formation during the
amplification reaction. The amplified PCR products were purified and
ligated into a pGEX-2T plasmid (Amersham Pharmacia Biotech, Uppsala,
Sweden) at the BamHI and EcoRI restriction sites.
The proper sequence of the resulting constructs was confirmed by
dideoxynucleotide sequencing prior to transformation into
Escherichia coli BL21(DE3)plysS for
isopropyl-1-thio- Construction of Hexahistidine-tagged (HIS-tagged) WT and
Non-folded Hybrid HMG-1 B-box Expression Vector--
The non-folded
hybrid B-box was constructed in the same manner as described above for
the correctly folded hybrid B-box but with slight modifications. The
DNA coding for an HMG-1 B-box lacking a 13-amino acid N-terminal
extended segment was constructed in a series of steps. 1) The
non-folded B-box w/o N-term (aa residues 88-100 of HMG-1) was
constructed by PCR amplification using the WT HMG-1 B-box PCR product
as the template with primers 6 and 7; 2) DNA coding for 13 amino acid
residues of the second A·T-hook motif of the HMG-I protein
(i.e. VPTPKRPRGRPK-K; corresponding to aa residues 51-62)
was constructed by PCR amplification of human HMG-I(7C) cDNA
template with primers 8 and 9. It should be noted that during this
construction the last lysine residue of this 13-amino peptide was
changed from the original glycine residue present in the native HMG-I
protein so that it now corresponds to the "consensus" A·T-hook
motif of the HMG-I(Y) family of proteins (8); 3) the non-folded hybrid
B-box was then constructed by annealing and extending the 13-amino acid
consensus A·T-hook motif with the non-folded B-box w/o N-term and
primers 8 and 7. As before, the 5' region of primer 6 is complimentary
to the 3' region of the consensus A·T-hook motif, and the 3' region
of the primer 9 is complimentary to the 5' region of the non-folded
B-box w/o N-term construct. Specific nucleotide base
mutations in the third base wobble position of the original HMG-I(7C)
cDNA clone that do not alter amino acid coding sequence were
engineered into the PCR primers to counteract primer-dimer formation.
The HIS-tagged WT HMG-1 B-box (corresponding to residues 88-164 of the
214-amino acid full-length WT HMG-1 protein; Ref. 21) was constructed by PCR amplification of human HMG-1 cDNA template using primers 10 and 7. In each case, dideoxynucleotide sequencing confirmed that DNA of
the correct nucleotide sequence had been produced. The amplified PCR
products were purified and ligated into the bacterial plasmid
expression vector pET-24b (Novagen, Madison, WI) between the
NdeI and XhoI restriction sites and transformed into E. coli BL21(DE3)plysS for production of recombinant
proteins as subsequently described below.
Expression and Purification of Recombinant Proteins--
GST
fusion WT and correctly folded hybrid B-box proteins were expressed and
purified as described by Read et al. (22), with slight
modifications. Prior to induction with 0.4 mM
isopropyl-1-thio-
HIS-tagged WT and non-folded hybrid B-box recombinant proteins were
then purified from inclusion bodies by the protocol described by Nagai
and Thogerson (23). Samples were solubilized to a 3 M final
concentration of guanidine HCl prior to Qiagen Ni(II)-NTA affinity
chromatography (Qiagen, Chatsworth, CA). A 100% linear gradient of
Buffer A (0.1 M NaPi and 0.01 M
Tris/HCl at pH 8.0) to Buffer B (0.1 M
NaPi, 0.01 M Tris/HCl, and 250 mM imidazole at pH 8.0) over 75 min was used to elute the
proteins. Dialysis of the protein solutions against 10 mM
Tris/HCl, pH 7.8, 1 mM DTT effectively removed
the imidazole.
Recombinant full-length human HMG-I(Y) and HMG-1 proteins were prepared
and chromatographically purified as described by Nissen et
al. (24) and by Hill et al. (25), respectively.
The size and purity of all of the recombinant proteins were verified by
SDS-polyacrylamide gel electrophoresis and matrix-assisted laser
desorption ionization mass spectrometry. In all cases, the proteins and
peptides used for experiments were purified to near homogeneity.
Concentration of the purified protein solutions was determined by
spectroscopy using extinction coefficients calculated from the Gill and
von Hippel formula (26) as utilized by the Genetics Computer Group
software program, PeptideSort. The extinction coefficient was
determined to be 10,870 liters/mol·cm for both the GST and
HIS-tagged WT and hybrid B-box proteins. The formula, as defined in
Ref. 26, is for an unfolded protein, yet the authors noted that in
general there is only a relatively small difference in the extinction
coefficient between an unfolded and folded protein.
Electrophoretic Mobility Shift Assays (EMSAs)--
The
double-stranded, A·T-rich, B-form DNA substrate used in EMSA
experiments was the well characterized 300-bp 3'-untranslated tail
region of the bovine interleukin-2 gene (BLT) and was prepared and used
as described previously (7, 11). Reactions containing 0.35 nM BLT DNA were incubated at room temperature from 10 to 20 min prior to loading and electrophoresed at 10 V/cm from 2 to 2.5 h at room temperature. Microccocal nuclease trimmed chicken erythrocyte
nucleosome core particles (CP) containing ~146 bp of DNA were
prepared as described in Ref. 27. EMSAs containing 0.5 ng (or 80 nM) CP DNA were incubated on ice prior to loading and were
electrophoresed at 4 °C for 2 h at 8 V/cm. In all experiments, the EMSA reactions were carried out in a total volume of 10 µl, with
protein binding buffer at a 1× concentration (10 mM
Tris/HCl, pH 7.8, 28 mM NaCl, 50 µg of bovine
serum albumin, 1 mM EDTA, 1 mM DTT, and 0.3 µg of dG-dC). BLT DNA EMSAs were loaded onto 6.5% (29:1), 0.25× TBE
native polyacrylamide gels, while the CP DNA reactions were run on 4%
(29:1), 0.25× TBE gels. The gels were dried and analyzed using
PhosphorImager analysis tools (Molecular Dynamics, Sunnyvale, CA).
DNase I Footprinting on BLT DNA--
DNase I footprinting
followed standard protocols (28, 29). 5 µM amounts of
either the correctly folded hybrid or the WT B-box proteins were bound
to BLT DNA (0.1 ng) in the footprinting reactions. Both the
concentration and time of digestion of DNase I were empirically
determined to obtain optimal results. Products of Maxam-Gilbert
chemical cleavage reactions (30) of BLT DNA served as reference
standards in the sequencing gels used in the footprinting experiments.
Circular Dichroism and Intrinsic Fluorescence Determinations of
Protein Structure--
In all circular dichroism analyses, the
absorption spectrum of recombinant protein preparations was measured at
least three times, with the average being used to calculate the molar
ellipticity. The background spectrum, consisting of buffer only, was
subtracted from each absorption spectrum. The correctly folded hybrid
B-box was measured under reducing conditions in 20 mM
KH2PO4, pH 6.0, and 1 mM
Tris-(2-carboxyethyl)-phosphine hydrochloride (Molecular Probes;
Eugene, OR) while the non-folded hybrid and both WT B-box proteins were
measured under non-reducing conditions in 20 mM KH2PO4, pH 6.0. Data were collected with
nitrogen purging on an AVIV 62DS CD instrument (AVIV Inc., Lakewood,
NJ). Intrinsic fluorescence measurements of the B-box proteins were
done on a Shimadizu 5300 spectrofluorometer (Shimadizu Corp., Kyoto,
Japan). Equal concentrations of proteins, solubilized in 10 mM Tris/HCl, pH 7.8, 1 mM DTT, were
used for the analysis. The excitation wavelength was 280 nm, and the
florescence emission was collected from 290 to 400 nm.
Rationale for Hybrid B-box Protein Construction and
Analysis--
For the reasons outlined above, the HMG-1 B-box peptide
was selected as the "highly structured" fusion partner for
incorporation into a "model" hybrid protein in which the N-terminal
segment of the B-box has been replaced with the A·T-hook DNA-binding
peptide motif (Fig. 1). As illustrated in
Fig. 1, the approximately 75 amino acid residues of the WT HMG-1 B-box
exist in solution as a twisted L-shaped structure consisting of three
stabilizing Expression and Analysis of Recombinant Wild Type and Hybrid B-box
Proteins--
Hybrid and WT B-box proteins were expressed both as GST
fusion and HIS-tagged recombinant proteins. The correctly folded hybrid and WT B-box proteins were expressed as soluble recombinant GST fusion
proteins, which were subsequently processed by cleavage with thrombin
to remove the GST moiety. To promote the formation of inclusion bodies,
prevent bacterial proteolytic degradation, and enhance the ease of
rapid purification, the non-folded hybrid and WT B-box proteins were
expressed with a HIS tag on their C termini.
As shown in Fig. 2, the GST-expressed WT and correctly folded hybrid
B-box proteins have slight differences in amino acid composition at
their N- and C-terminal ends relative to the HIS-tagged WT and
non-folded hybrid B-box proteins. After thrombin cleavage and
subsequent purification, both the GST-derived WT and correctly folded
hybrid B-box proteins are 84 amino acids in length (Fig. 2A,
rows b and c). In both of these
isolated recombinant proteins, eight amino acids (i.e. GS on
the N terminus and EFIVTD on the C terminus) are derived from the
pGEX-2T plasmid expression vector. The HIS-tagged proteins are similar
in length at 83 amino acids but have six histidine residues attached on
their C termini (Fig. 2A, rows a and
d). Regardless of these minor differences, the various
recombinant WT B-box proteins had similar biophysical properties and
also behaved identically in all experimental assays (data not shown),
indicating that their slight differences in amino acid composition did
not have any observable influence on their substrate-binding properties
under our experimental conditions. It can therefore be argued that any
biophysical differences that are observed between the HIS-tagged,
non-folded B-box hybrid and the correctly folded recombinant B-box
hybrid are due to variations in the state of folding of their
respective peptide domains and are unrelated to their mode of preparation.
As an experimental control for the correctly folded B-box proteins,
site-specific mutations were introduced into the hybrid B-box that
interfered with the protein's ability to correctly fold into its
normal tertiary configuration. The engineered differences in this
non-folded hybrid B-box, as compared with the correctly folded hybrid,
are two lysine residues that have been introduced at the C-terminal end
of the A·T-hook motif (aa 12 and 13) that replace both a
serine and a highly conserved alanine residue (32) (Fig. 2B;
compare rows c and d in Fig.
2A). The positioning of these two positively charged lysine
residues, in close proximity to the hydrophobic hinge region of the
B-box (Fig. 1), disrupts the hydrophobic packing interactions and
thereby prevents proper tertiary folding of the mutant protein. This
predicted structural disruption was confirmed by subsequent structural
analyses (see below).
Circular Dichroism and Intrinsic Florescence Structural Analysis of
Recombinant Hybrid and WT B-box proteins--
Circular dichroism (CD)
gives information concerning the relative amount of secondary structure
in a molecule and was used to verify that both the correctly folded
hybrid and the WT B-box proteins contained their normal
In contrast to the situation for the properly folded recombinant
proteins noted above, the CD spectrum at 25 °C for the non-folding hybrid B-box protein (Fig. 3B) is very similar to the random
coil profile of proteins denatured at 80 °C (Fig. 3, A
and B). Consistent with this interpretation, the IF spectrum
for the non-folded hybrid B-box (Fig. 3D) is quite different
from that of properly folded B-box proteins. The non-folded hybrid
B-box exhibits a red shift of nearly 20 nm for the wavelength showing
maximum fluorescence (350 nm versus 330 nm) and a
significant decrease in the overall quantum yield of fluorescence
relative to the three other B-box proteins (Fig. 3, C and
D). In combination with the CD data, the IF measurements
indicate that the non-folded hybrid B-box is not forming Hybrid B-box Proteins Preferentially Bind to A·T-rich BLT
DNA--
Fig. 4 shows a comparison of various recombinant proteins
binding to a well characterized A·T-rich duplex DNA substrate, BLT, the 3'-untranslated tail region of the bovine interleukin-2 cDNA (7, 11). As noted previously, whereas HMG-I(Y) is known to specifically
bind to the A·T-rich regions of the BLT DNA, both the full-length
HMG-1 protein and isolated WT B-box peptides bind only nonspecifically
to B-form DNA substrates. Fig. 4A illustrates that the
full-length HMG-I(Y) protein forms a number of specific protein-DNA
complexes with BLT DNA as a function of increasing concentrations of
protein. In contrast, both the full-length HMG-1 protein (Fig.
4B) and the WT B-box (Fig. 4D) bind only
nonspecifically to the BLT DNA, as evidenced by the pronounced smearing
of the protein-DNA aggregates in both gels. In this context, it should be noted that the apparent bands seen in the 4.5 µM and
5.5 µM lanes of Fig. 4B are not
specific HMG-1-DNA complexes but rather are nonspecific saturation
aggregates trapped in and near the sample loading wells. Importantly,
the correctly folded hybrid B-box (Fig. 4C) forms a number
of discrete protein-DNA complexes reminiscent of the specific complexes
formed by the full-length HMG-I(Y). It is readily apparent that the
specific protein complexes seen in Fig. 4 (A and
C) are quite different from the nonspecific complexes formed
by either the full-length HMG-1 or the WT B-box proteins. It should be
noted, however, that there are several orders of magnitude difference
in the binding affinity of HMG-I(Y) and the correctly folded hybrid
B-box protein for BLT DNA. This difference in binding affinity is at
least partially explained by the differing number of DNA-binding motifs
present in the two proteins. While the full-length HMG-I(Y) protein,
which has three independent A·T-hook motifs that interact
cooperatively with A·T-rich substrates (8, 9), binds BLT DNA in the
nanomolar range (Fig. 4A), the correctly folded hybrid B-box
protein, which has only a single A·T-hook motif and consequently has
a lower affinity for this substrate (8), binds in the micromolar range.
In addition, constraint of the normally flexible A·T-hook motif in
the confines of the rigid B-box configuration likely contributes to the
reduced binding affinity of the correctly folded hybrid (9). Consistent with this interpretation, the non-folded hybrid B-box, as shown in Fig.
4E, also forms discrete complexes with BLT DNA at a higher affinity (in the submicromolar range) than the correctly folded hybrid
B-box. This is reasonable considering that the A·T-hook motif in the
non-folded hybrid B-box is not confined by a rigid structural
scaffolding and is therefore quite flexible, similar to the situation
in the tightly binding HMG-I(Y) protein itself (9). Nevertheless, both
hybrid proteins are able to form specific protein-DNA complexes,
suggesting that the ability to bind specifically to A·T-rich
sequences has been conferred to the fusion proteins by the A·T-hook
DNA binding motif. Importantly, these data demonstrate that the
functional A·T-hook peptide structure is not seriously compromised by
the presence of the rigid structural elements of the HMG-1 B-box in the
correctly folded hybrid protein.
A series of DNA competition experiments were performed to verify, by an
independent technique, that the correctly folded hybrid B-box protein
is recognizing the narrow minor groove structure associated with
stretches of A·T-rich DNA. Fig. 5 shows
the results of experiments in which the correctly folded hybrid B-box
protein was incubated with radiolabeled BLT DNA in the presence of
increasing concentrations of one of two different non-labeled DNA
competitors: dG-dC, a nonspecific competitor DNA, or dI-dC, a specific
competitor for A·T-rich DNA sequences. As seen in Fig. 5
(A and B), when the correctly folded hybrid B-box
is bound to labeled BLT DNA without any added non-labeled competitor
there is smearing, characteristic of nonspecific protein-DNA
interactions. However, as shown in Fig. 5A, in the presence
of low concentrations of dG-dC (e.g. 0.1 µg/reaction), these nonspecific protein-DNA interactions are abated, and it is possible to resolve both a specific protein-DNA complex and the unbound free DNA on the gel. Furthermore, as expected with any nonspecific competitor, at considerably higher concentrations of dG-dC (e.g. 0.5 µg/reaction) the specific
hybrid B-box-DNA complex is also eliminated (Fig. 5A). On
the other hand, in titration reactions containing increasing
concentrations of unlabeled dI-dC that compete with A·T-DNA for
binding (Fig. 5B), no specific protein-DNA complexes are
observed under any conditions. Important for the interpretation of
these competitions is the fact that dI-dC has been shown to effectively
compete with the HMG-I(Y) protein for binding to the A·T-rich
sequences present in BLT DNA and other substrates (8,
10).2 These results suggest
that the correctly folded hybrid B-box protein recognizes the
structural characteristics of a narrow minor groove such as that
associated with A·T-rich DNA.
Correctly Folded Hybrid B-box Proteins Footprint to A·T-rich
Sequences--
Fig. 6 shows the results
of DNase I footprinting experiments in which a direct comparison was
made between the binding of equimolar concentrations (indicated by the + lanes) of the correctly folded hybrid B-box protein
(panel A) and the GST-expressed WT B-box protein
(panel B) on A·T-rich BLT DNA. As indicated by
the vertical lines adjacent to the + lane in panel A, the correctly folded
hybrid B-box protein clearly shows protection of several A·T-rich
regions in BLT DNA, relative to the nuclease cleavage of naked DNA
shown in the Both HMG-I(Y) and Hybrid B-box Proteins Bind to Nucleosome Core
Particles--
It is well established that, in chromatin, the HMG-1
protein binds to the DNA linker region separating adjacent nucleosomes but does not bind in vitro to isolated nucleosome core
particles that lack linker DNA (1). In marked contrast, as illustrated in Fig. 7A, HMG-I(Y) can form
up to four or more specific complexes with the random sequence
nucleosome core particles isolated from chicken erythrocytes. These
data are consistent with previous observations (27) and are thought to
be a consequence of the A·T-hook motifs of the HMG-I(Y) protein
recognizing certain distorted structural features of the DNA as it
wraps around the histone octamer (27, 29). In a similar fashion, as
indicated by the arrows in Fig. 7B, the correctly
folded hybrid B-box also forms a number of distinct complexes with
nucleosome core particles, although with a distinctly reduced binding
affinity relative to the HMG-I(Y) protein (i.e. micromolar
versus nanomolar). As mentioned previously, this reduced
core particle binding affinity is most likely a consequence of there
being only a single A·T-hook peptide sequence in the hybrid protein
combined with a reduced flexibility of this peptide when it is
incorporated into the highly structured HMG-1 B-box scaffold.
Significantly, neither the full-length HMG-1 protein (Fig.
7C) nor the WT B-box peptide (Fig. 7D) binds to nucleosome core particles under these same reaction conditions. Together these data clearly demonstrate that the single A·T-hook motif in the correctly folded hybrid B-box protein has imparted important HMG-I(Y)-like chromatin binding properties to the chimeric protein.
In addition to chromosomal translocation of their A·T-hook
DNA-binding motifs (14), constitutive transcriptional overexpression of
the genes coding for the HMG-I(Y) family of nonhistone chromatin proteins has also been associated with a wide variety of tumor types in
humans and other mammals. Given the fact that the HMG-I(Y) proteins are
in vivo regulators of gene transcriptional activity (1) and
also are involved in cellular growth regulation (35-37), it comes as
no surprise that overexpression, mis-regulation, or chromosomal
translocation of the DNA-binding regions of the HMG-I(Y) genes
significantly contributes to processes such as oncogenic transformation, increased tumor metastatic potential, and overt neoplastic malignancy. What remains unclear, however, is the precise molecular role(s) played by the HMG-I(Y) proteins, or their
derivatives, in these tumorigenic processes.
Considerable uncertainty surrounds the possible physiological roles, if
any, played by the translocation-derived, A·T-hook-containing, chimeric fusion proteins associated with a wide variety of benign tumors and overtly malignant cancers (Table
I). Here, employing domain-swap
experiments in a well characterized in vitro model protein
system, we have demonstrated that the A·T-hook motif of the HMG-I(Y)
protein family maintains its A·T-DNA-binding, and its nucleosome core
particle-binding, specificity in both an unstructured non-folded hybrid
protein and in a structurally rigid correctly folded hybrid protein.
These findings are important because they strongly suggest that
A·T-hook-containing chimeric tumor proteins likewise retain many of
the substrate-binding properties of the HMG-I(Y) protein regardless of
whether or not these hybrids are folded in vivo into a
distinct tertiary configuration. Although the structural
characteristics of the folded ectopic peptide partners fused to
A·T-hooks are unlikely to radically alter the substrate binding
specificity of the A·T-hook motifs in vivo, they may, nevertheless, considerably reduce the affinity of the hybrid proteins for their substrate binding sites. The conferral of HMG-I(Y)-like substrate binding characteristics to chimeric tumor proteins in vivo thus provides a potential focus for the development of a new
category of cancer therapeutic drugs aimed at selectively disrupting
such aberrant protein-DNA interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' Sequence of Polymerase Chain Reaction (PCR) Primers
used for Expression Vector Construction--
Sequences are as
follows: primer 1, ATGGATCCAAGGACCCTAATGCA; primer 2, TGAATTCTTTTGCACGGTATGC; primer 3, CGTGGTCGACCTTCGGCATTCTTC; primer 4, TGGATCCCCAACACCTAAG; primer 5, GAAGAATGCCGAAGGTCGACCACGAGG; primer 6, CGACCCAAGAAGTTTTTCCTATTCTGC; primer 7, TTCTCGAGTTTAGCTCGATA; primer 8, AAATAACTCATATGGTTCCAACACCT; primer 9, GAATAGGAAAAACTTCTTGGGTCGGCC; primer 10, TGGAATTCCATATGTTCAAGGATCCC.
-D-galactopyranoside-induced production
of recombinant GST fusion proteins.
-D-galactopyranoside, the LB culture
was heat-shocked at 42 °C for 5 min to induce chaperone proteins.
After heat shock, the cultures were grown for an additional 3 h
before processing. The cells were sonicated, and Nonidet P-40 was added
to a final concentration of 1%. After centrifugation to pellet
cellular debris, the GST fusion protein containing supernatant was
incubated with glutathione-Sepharose 4 beads (Amersham Pharmacia Biotech) with shaking at 4 °C for several hours. The beads with the
GST fusion proteins attached were washed extensively with 300 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM DTT wash solution (22). The wash solution was made 2 mM with respect to CaCl2 and MgCl2,
and the GST fusion proteins attached to the beads were cleaved with
thrombin overnight at 4 °C. The beads were centrifuged, the
supernatant containing the free recombinant protein cleaved of its GST
moiety was removed, and fresh wash solution, CaCl2, MgCl2, and thrombin were incubated again with the beads
overnight at 4 °C. This procedure was repeated until
SDS-polyacrylamide gel electrophoresis showed few, or no, GST fusion
proteins remaining attached to the beads. The released recombinant
proteins were further purified away from the thrombin by reverse-phase
high pressure liquid chromatography. The purified recombinant proteins were lyophilized and reconstituted in 10 mM
Tris/HCl, pH 7.8, 1 mM DTT.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices and an "extended" N terminus peptide (19,
20). The extended N terminus is the region of the B-box that is
primarily responsible for making initial minor groove contacts with DNA
substrates (22). Importantly, this N-terminal peptide shares a common
tetrapeptide sequence (P-K-R-P) with the A·T-hook DNA-binding motif
(Figs. 1 and 2) and has three similarly
spaced proline residues (all in the trans configuration) at
aa positions 4, 7, and 11 (9, 12). Such similarities suggest that these
two peptide segments may have evolved from a common evolutionary
ancestral sequence (1). Given these common features in their respective
DNA-binding regions, it is not surprising that both the WT HMG-1 and
the HMG-I(Y) proteins share similar abilities to bind to the DNA minor
groove, to selectively bind to bent, supercoiled, or distorted DNA
substrates and to selectively bind to non-B form DNA structures such as
synthetic four-way junctions (1, 25). Nevertheless, there are also a
number of significant differences between these two proteins that can
be exploited to both qualitatively and quantitatively distinguish
between them, and, therefore, characterize the substrate binding
characteristics of an artificially produced recombinant hybrid
A·T-hook/B-box protein (1). For example, whereas HMG-1 binds to
double-stranded DNAs in a sequence-independent manner, the HMG-I(Y)
proteins specifically bind to A·T-rich substrates. Also, whereas the
HMG-I(Y) proteins can bind to nucleosome core particles (27, 29, 31),
the HMG-1 protein cannot, instead preferentially binding to the linker
DNA between adjacent nucleosomes in chromatin (1).
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Fig. 1.
Peptide domain-swap strategy for exchanging
the extended N-terminal region of the HMG-1 B-box with the second
A·T-hook motif from HMG-I(Y). The sequences of the two 10-aa
peptides involved in the exchange are shown. The horizontal
cross-hatched bar between the sequences indicates
the area of the palindormic "core" residues of the A·T-hook
peptide with a glycine (G) at its center. The
italicized letters denote the identical spacing
of the trans configuration proline residues in the two
peptides. Only 5 of the 10 aa from the second (II) DNA-binding motif of
HMG-I(Y), that correspond to the "core" region (PRGRP), are modeled
with and without DNA to illustrate the planar, crescent-shaped
structure formed upon substrate interaction (modeling done using
Rasmol; Ref. 40). The three-dimensional L-shaped structure of the HMG-1
B-box peptide is based on the NMR data of Weir et al. (20)
and shows the position of the highly conserved tryptophan residue in
the hydrophobic hinge region of the correctly folded molecule. In
addition, key hydrophobic residues in the C-terminal third helix are
modeled to illustrate the existence of a hydrophobic interface formed
with the extended N terminus. The model of the B-box was generated
using MOLSCRIPT (41).
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Fig. 2.
Comparison of the correctly folded and
non-folded hybrid B-box proteins with the WT HMG-1 B-box protein.
The WT B-box corresponds to residues 89-164 of the 214-amino acid
full-length HMG-1 protein. Panel A, the sequences
of the hexahistidine-tagged (HIS-tagged) WT (a) and
non-folded hybrid (d) proteins are compared with those of
the GST-expressed WT (b) and correctly folded hybrid B-box
(c) proteins. Panel B, a more detailed
comparison of the N-terminal regions of the correctly folded and the
non-folded hybrid B-box proteins is shown here. The box
indicates the location of two genetically engineered lysine residues at
positions 12 and 13 in the non-folded hybrid that are responsible for
the structural disruption the domain.
-helical
secondary structural elements. In addition, CD analysis verified the
predicted structural disruption of the non-folded hybrid B-box. As
shown in Fig. 3 (A and
B), the spectra at 25 °C for both the WT B-box and
correctly folded hybrid B-box proteins derived from GST fusion products resemble those previously reported for correctly folded HMG box peptides (22, 33). The CD data were deconvoluted (34) to yield
approximate percent values of
-helix,
-turn, and random coil for
the WT and correctly folded hybrid proteins that, within experimental
error, are quite similar to each other (see Fig. 3, A and
B). Fig. 3 (A and B) also demonstrates
that upon heating to 80 °C, the CD spectra for both proteins shift
toward a shape characteristic of an irregular or random coil. The
HIS-tagged WT B-box protein was also examined using CD at both 25 °C
and 80 °C and is nearly identical to the hybrid and WT proteins
derived from GST fusion products (data not shown). Together, these data sets indicate that all three of these recombinant proteins have refolded properly at 25 °C and contain
-helical folds
characteristic of a WT B-box domain (19, 20, 22). The slight
differences in the shape of the two CD spectra for the correctly folded
hybrid and the WT B-box proteins at both 25 and 80 °C (Fig. 3,
A and B) are attributable to slight differences
in buffer conditions (see "Materials and Methods"). The intrinsic
fluorescence (IF) measurements shown in Fig. 3 (C and
D) likewise confirm that the tertiary configurations of the
correctly folded hybrid and the two WT B-box proteins are very similar,
if not identical. These data indicate that the tryptophan residues in
the hydrophobic hinge regions of these three B-box peptides are equally
protected from solvent accessibility. Importantly, the IF spectra for
all three of the proteins are also almost superimposable, exhibiting similar quantum yields and an emission maxima close to 330 nm (Fig.
3, C and D). If the
N-terminal extended peptide region (which contains the A·T-hook) were
introducing a substantial deformation into the overall
-helical
structure of the chimeric B-box peptide, the result would be a marked
difference in the IF emission spectra of the hybrid derived from GST
fusion products relative to that of the correctly folded WT B-boxes. As
can be seen in Fig. 3 (C and D), this is not the
case. Therefore, based on both the CD and IF measurements, we conclude
that, under our experimental conditions, the recombinant folded hybrid
and both of the WT B-box proteins are correctly folded into a tertiary
structure characteristic of a WT HMG-1 B-box protein.
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Fig. 3.
CD and IF measurements of B-box
proteins. Panel A, the structural elements
of the WT B-box protein (GST-expressed), as monitored by CD, were
measured at 25 °C and 80 °C. Note the loss of -helical
structure when the protein is denatured at 80 °C. Panel
B, the correctly folded hybrid B-box protein exhibits a
similar CD pattern to that of the WT B-box protein at both 25 and
80 °C. However, at 25 °C, the non-folded hybrid B-box protein has
a CD spectrum similar to that seen for the denatured correctly folded
hybrid at 80 °C. Panel C, IF measurements
(excitation = 280 nm; emission = 290-400 nm) confirmed the
structural similarities between the correctly folded hybrid and the WT
B-box proteins. Panel D, the red shift and
decreased quantum yield of the IF spectrum of the non-folded hybrid
(HIS-tagged), resulting from solvent accessibility to the hydrophobic
pocket of the domain, relative to the WT B-box protein (also
HIS-tagged), confirms a lack of proper folding in this chimeric
protein.
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Fig. 4.
Both HMG-I(Y) and hybrid B-box proteins
specifically bind to A·T-rich B-form DNA. EMSAs were performed
using various recombinant proteins and the A·T-rich BLT DNA as a
substrate. BLT DNA is the 300-bp 3'-untranslated tail region
of the bovine interleukin-2 cDNA, which contains multiple
A·T-rich binding sites for the HMG-I(Y) protein (7, 11).
Panel A, full-length wild-type HMG-I(Y) protein.
Panel B, full-length wild-type HMG-1 protein.
Panel C, correctly folded hybrid B-box protein.
Panel D, wild-type B-box protein.
Panel E, non-folded hybrid B-box protein. Note
that the specific BLT-binding characteristics of the HMG-I(Y) protein
(A) are mimicked in the both the correctly folded
(C) and non-folded (E) hybrid B-box
proteins. In contrast, both the full-length HMG-1 protein
(B) and WT B-box protein (D) bind only
nonspecifically as indicated by smearing.
-helical
secondary structural elements to the same degree as the correctly
folded hybrid or WT B-box proteins. These data also demonstrate that
the non-folded hybrid B-box exhibits physical characteristics more
closely resembling those of denatured, rather than native, proteins.
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Fig. 5.
Competition EMSA demonstrate that the
correctly folded hybrid B-box recognizes the narrow minor groove of
DNA. Increasing concentrations of various unlabeled
double-stranded B-form competitor DNAs were added to reaction mixtures
containing a fixed amount of the correctly folded hybrid B-box (1.3 µM) and labeled BLT DNA (0.35 nM).
Panel A, competition using the dG-dC competitor
DNA. Panel B, competition using dI-dC competitor.
Without competitor DNA, the proteins bind nonspecifically to BLT as
evidenced by smearing. Low concentrations of dG-dC competitor DNA allow
a specific protein-DNA complex to be resolved between the hybrid and
A·T-rich DNA. However, dI-dC, whose narrow minor groove mimics that
of A·T-rich DNA, effectively competes the correctly folded hybrid
from the labeled substrate at low concentrations. These results suggest
that the correctly folded hybrid protein is recognizing the structure
associated with the narrow minor groove of its DNA targets.
lane. In contrast, the WT B-box protein does not show protection of the substrate relative to the
lane (Fig. 6B). In fact, the pattern of DNase I
protection for the correctly folded hybrid B-box protein to these
A·T-rich regions in the BLT DNA is similar, if not identical, to the
previously published binding sites for the HMG-I(Y) protein on this
same substrate (7, 11). These footprinting results, combined with the
previously described results from the EMSA and substrate competition experiments, conclusively demonstrate that the specificity of the
correctly folded hybrid B-box protein has been altered to closely
resemble that of the HMG-I(Y) protein for A·T-rich regions of B-form
DNA.
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Fig. 6.
DNase I footprinting demonstrates that the
correctly folded hybrid B-box protein specifically binds to A·T-rich
sequences, whereas the WT B-box protein does not. Panel
A, correctly folded hybrid B-box protein (5 µM) footprinted on BLT DNA. The sites of specific
protection of A·T-rich sequences by the bound protein are indicated
by the vertical lines adjacent to the panel. Panel
B, wild-type B-box protein (5 µM) incubated
with BLT DNA. The correctly folded hybrid B-box protein (A)
shows a similar pattern of protection of the A·T-rich of the BLT
substrate to that previously reported for the full-length HMG-I(Y)
protein on this DNA (13). However, as expected, the WT B-box does not
show specific protection of any sequences relative to naked DNA. The + indicates lanes in which protein samples were incubated with the BLT
DNA whereas the indicates lanes with naked DNA alone. Lanes in
which the BLT DNA has been chemically cleaved by Maxam-Gilbert "G"
and "G+A"reactions are included in each panel as a reference.
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Fig. 7.
Both HMG-I(Y) and the correctly folded hybrid
B-box proteins bind specifically to isolated nucleosome core
particles. EMSAs were used to monitor the interaction of various
recombinant proteins with nucleosome core particles (146 bp) isolated
from chicken red blood cells. Panel A, HMG-I(Y)
protein. Panel B, correctly folded hybrid B-box
protein. Panel C, full-length HMG-1 protein.
Panel D, wild-type B-box protein. Note that both
the HMG-I(Y) protein (A) and the correctly folded hybrid
B-box protein (B) formed a number of specific complexes with
the isolated nucleosomes, whereas neither the HMG-1 protein
(C) nor the wild-type B-box (D) protein bound to
such core particles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
A · T-hook-containing human fusion proteins
The present results also raise intriguing questions about the structural features of both the B-box and the A·T-hook that permit the fusion product of these two very different peptides to form a correctly folded chimeric protein, which, on the one hand, has the rigid tertiary structure of the B-box of the HMG-1 protein family and yet, on the other, retains the substrate binding specificity of the HMG-I(Y) protein family. Considerable insight into this problem comes from analysis of computer modeling studies based on the known structures of both the HMG-box and A·T-hook peptides as they exist either free in solution (12, 19, 20, 38) or as co-complexes with DNA substrates (9, 39).
Solution NMR studies of a co-complex of the DNA-binding domains of HMG-I(Y) with a synthetic substrate indicate that the A·T-hook undergoes a disordered-to-ordered structural transition upon binding that is necessary for selective association with the minor groove of A·T-rich sequences. As evidenced by the results presented here, the positioning of the A·T-hook in the extended peptide region of the correctly folded hybrid B-box is not likely to significantly disrupt this critical substrate-associated structural transition. Indeed, molecular modeling studies predict that in the correctly folded hybrid both the peptide backbone and the side chains of the arginine residues in the palindromic "core" sequence of the A·T-hook motif (i.e. PRGRP; Fig. 1) can assume the physical conformation necessary for interactions with the minor groove of A·T-DNA, thus conferring the appropriate substrate specificity on the chimeric B-box protein (data not shown). On the other hand, molecular modeling also indicates that the peptide backbone and the side chains of the lysine and arginine residues in the N-terminal extended domain of the WT B-box (19) are unable to assume such a physical configuration thus restricting the ability of the WT B-box to specifically bind to A·T-DNA sequences.2 Together these results provide a structural rationale for explaining the substrate binding differences between these otherwise quite similar proteins.
There are numerous biological implications from the present work with
regard to the possible etiology and maintenance of human tumors
associated with chromosomal translocations involving the A·T-hook
motif. The first, and most obvious, of these is that many of the
chimeric A·T-hook-containing fusion proteins probably have acquired
HMG-I(Y)-like DNA and nucleosome binding properties in vivo,
an inference supported by observations made with the translocated
A·T-hook motifs of the mixed lineage leukemia gene (17). But perhaps
just as importantly, the present results are consistent with the idea
that these novel tumor-associated hybrid fusion proteins may have also
lost other properties that are necessary for normal in vivo
functioning of the HMG-I(Y) proteins. Included among these likely
aberrant properties of the chimeric proteins are: weakening of the
normally high affinity binding of the A·T-hook motifs to A·T-rich
DNA substrates, loss of cell cycle-regulated transcriptional expression
of the hybrid gene, loss of a linkage of expression of the hybrid gene
to the differentiated state of the cell, loss of the ability of the
hybrid protein to make specific associations with other proteins, loss
of specific sites for secondary biochemical modifications in the
hybrids, loss of normal mechanisms regulating stability
and/or translational efficiency of the hybrid transcripts,
alterations in the stability and/or intracellular
localization of the hybrid proteins, as well as others. Thus,
overexpression of A·T-hook-containing, but otherwise defective,
hybrid fusion proteins could be expected to lead to abnormal gene
transcriptional expression, as well as major alterations in chromatin
structure, in tumor cells. Speculation as to the in vivo
role played by these chimeric proteins often tends to focus on the type
of protein partner fused to the A·T-hook motif when, in fact, the
most important aspect of these hybrid tumor proteins may be the
abnormal functioning of A·T-hook motifs themselves. Thus, processes
related to the A·T-hook motif such as constitutive overexpression,
and/or deregulation of expression of A·T-hook-containing proteins are likely to be at least as important as the nature of the
ectopic fusion partner per se. Future characterization of
the structure and DNA-binding properties of individual chimeric proteins in specific tumors should provide more insight into the role
and functionality of the A·T-hook motif in tumorigenesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. David Baker and Dr. Michael A. Kennedy for use of their circular dichroism instruments, Mark Nissen for his advice, and Gerhard Munske for the synthesis of primers used in PCR.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-46352 (to R.R.).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.: 509-335-1948; Fax: 509-335-9688; E-mail: reevesr{at}mail.wsu.edu.
2 G. C. Banks, B. Mohr, and R. Reeves, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HMG, high mobility group nonhistone chromatin proteins; aa, amino acid(s); BLT, DNA from the 3'-untranslated tail region of the bovine interleukin-2 cDNA; CD, circular dichroism; CP, nucleosome core particle; EMSA, electrophoretic mobility shift analysis; GST, glutathione S-transferase; A·T-hook, the highly conserved DNA-binding domain of the HMG-I(Y) family of nonhistone proteins; IF, intrinsic fluorescence; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; WT, wild type; DTT, dithiothreitol; bp, base pair(s).
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REFERENCES |
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