From the Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260-1300
Received for publication, December 12, 2002, and in revised form, January 28, 2003
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ABSTRACT |
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The sequence of non-contacted bases at the center
of the 434 repressor binding site affects the strength of the
repressor-DNA complex by influencing the structure and flexibility of
DNA (Koudelka, G. B., and Carlson, P. (1992) Nature
355, 89-91). We synthesized 434 repressor binding sites that differ in
their central sequence base composition to test the importance of minor
groove substituents and/or the number of base pair hydrogen bonds
between these base pairs on DNA structure and strength of the
repressor-DNA complex. We show here that the number of base pair
H-bonds between the central bases apparently has no role in determining
the relative affinity of a DNA site for repressor. Instead we find that
the affinity of DNA for repressor depends on the absence or presence the N2-NH2 group on the purine bases at the binding site
center. The N2-NH2 group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by
decreasing the intimacy of the specific repressor-DNA contacts, while
increasing the reliance on protein contacts to the DNA phosphate
backbone. Thus, the presence of an N2-NH2 group on the
purines at the center of a binding site globally alters the precise
conformation of the protein-DNA interface.
It is well established that the sequence-specific binding of
proteins to DNA involves specific contacts between DNA bases and
protein side chains in a process known as "direct-readout." The
precise alignment of DNA and protein can be specifically modulated by
the sequence of DNA bases in the binding site that are not directly
contacted by the protein. This phenomenon is known as indirect readout.
In indirect readout, the affinity or specificity of a protein-DNA
complex depends on sequence-dependent alterations in the
conformation and/or conformational flexibility of the noncontacted bases in the DNA site.
Since sequence-dependent DNA structural differences play a
role in mediating protein-DNA complex formation, this implies that a
protein binds only to a distorted DNA conformation. Noncontacted bases
may be envisioned to affect the formation of a protein DNA complex in
any of three ways: 1) altering the deformability of DNA; 2) altering
the structure of the DNA in the bound complex, thereby changing the
strength of particular protein-DNA contacts; or 3) altering the
structure of the unbound DNA, thereby eliminating or requiring the
imposition of energetically costly large-scale DNA conformational
changes. Although indirect readout is part of the sequence recognition
mechanism of many sequence-specific DNA binding proteins, little is
known about the physical basis of how base sequence and/or the
functional groups on the bases contribute to the sequence dependence of
such DNA deformations. In addition, the mechanisms by which
sequence-dependent differences in DNA conformation
influence the strength or specificity of protein-DNA complexes is also unclear.
434 repressor does not contact the functional groups on bases at the
center of its binding site (1, 2; see also Fig. 1). Nonetheless, changing the sequence of these bases remarkably influences the affinity
of DNA for repressor (3). Biochemical and crystallographic studies show
that, in complex with protein, the noncontacted bases at the center of
the 434 binding site are overwound and the minor groove in this region
of the binding site is narrower than in canonical B-DNA (1, 3-6).
Binding sites with higher intrinsic twists bind 434 repressor with
higher affinity than do those with lower twist (5, 6). Also, sites that
bear central sequences or modifications that increase twisting
flexibility bind repressor with higher affinity than those that do not
(3, 7). Hence, the sequence of the noncontacted central bases in the
434 binding site affects its affinity for repressor by regulating both
the structure of the unbound binding site and the ease with which the
site adopts the overtwisted conformation necessary to bind repressor.
Despite knowing how sequence-dependent differences in DNA
twist influence the affinity of a binding site for repressor, we still
do not precisely know how sequence determines DNA twist and/or twisting
flexibility. Two observations focus our attention on the
N2-NH2 group of the purine bases present at the center of
the 434 repressor binding site (positions 6-9). First, introducing this group at the center of the binding site by changing the
noncontacted central base sequences from one that is A/T-rich to one
that is G/C-rich decreases the affinity of the DNA for repressor by
60-fold. Second, binding sites bearing I·C base pairs at the central
positions bind repressor as well as do binding sites bearing A/T-rich
sequences at their centers. Inosine is identical to guanine, except
that it lacks the N2-NH2 group on the minor groove surface
of the base and thereby resembles A·T base pairs in the minor groove.
Hence, the presence of the N2-NH2 group appears to decrease
the affinity of a binding site for 434 repressor.
How might the N2-NH2 group exert this deleterious effect on
affinity? Here we test two ideas. First, since purine bases bearing an
N2-NH2 group form a third base pair hydrogen bond between
the base it is possible that this "extra" hydrogen bond renders
binding sites bearing G/C base pairs at their center less readily
deformable than those containing A/T (or I/C) pairs at these positions.
Alternatively, the bulky N2 amino group of guanine may sterically or
electrostatically oppose the repressor-induced DNA overwinding of this
region of the DNA.
DNA Synthesis and Preparation--
All oligonucleotides used in
the studies were purchased from the CAMBI Nucleic Acid Facility
(University at Buffalo). The binding sites were synthesized as a
self-complimentary single DNA strand 50 bases long (8). These
single-stranded DNAs were purified from denaturing gels and resuspended
in buffer (25 mM NaPO4, pH 6.8, and 25 mM NaCl). All purified DNAs were heated to 70 °C and
allowed to self-anneal by cooling slowly to room temperature.
Filter Binding Assays--
Approximately 0.5 µg of each
hairpin was 5' end-labeled by incubating the DNA with 20 µCi of
[ Circular Dichroism--
All circular dichroism
(CD)1 measurements were
performed using a Jasco J715 spectropolarimeter (Pharmaceutical
Sciences Instrumentation Facility, University at Buffalo). Data were
acquired from 220-310 nm at 0.5-nm intervals, scanned at 20 nm/min.
The final spectrum represents an average of three separate scans. CD
measurements for each sample were performed at 2 or 50 mM
concentration to ensure that CD features were not derived from
oligomerization of the DNA.
Hydroxyl Radical Cleavage--
Hydroxyl radical cleavage
experiments were performed essentially as described previously (10).
Cleavage of DNA in the absence of repressor was performed to give, on
average, one cleavage per DNA molecule. Following cleavage, the
reaction product was phenol/chloroform-extracted and fractionated on
12% denaturing polyacrylamide gels and the gels exposed to an imaging
plate. Quantitation of cleavage intensity was performed using Image
QuantTM software. Each experiment was repeated two to five
times. To compare results between individual experiments, band
intensities within the binding site were normalized to control bands
flanking the site. The standard deviations of band intensities varied
by less than 20% between identical experiments.
Affinity of Synthetic 434 Repressor Binding Sites for 434 Repressor
Depends on Base Substituents at Positions 7 and 8--
434 repressor
binding sites that differ in their central sequence base composition
were synthesized to test the importance of minor groove substituents
(the N2-NH2 group) and/or hydrogen bonding in binding
affinity to repressor (see Fig. 2). In a design that mimics that used
in studies of trp repressor binding to DNA (8), the 434 binding site
was embedded within a 50-base "hairpin". The affinity of 434 repressor for an oligonucleotide site bearing T·A bases at positions
7 and 8 is dramatically higher than that for the site bearing G·C
bases at these positions (Fig. 2). These findings are in agreement with
our previous measurements using 434 binding sites embedded in longer
DNA fragments (3, 5, 7). Hence, embedding the 434 binding site in a
short hairpin does not affect the DNA sequence recognition by 434 repressor.
Substituting either diaminopurine (DAP) or 2-amino purine (AP) for
adenine at position 8 of the binding site decreases the affinity of the
DNA for repressor between 21 and 42-fold (Fig. 2) at 150 mM
KCl. Since both of these substitutions introduce an NH2
group at position 8, this finding shows that the presence of an
N2-exocyclic amino group in the minor groove at the center of the
binding site is deleterious for the affinity of repressor for DNA.
Consistent with this finding, removing the N2-NH2 group from the minor groove by substituting inosine for guanine at position 7 increases the affinity of repressor for DNA by nearly a factor of
104 at 150 mM KCl.
When present on the purines at positions 7 or 8, the N2-NH2
group projects from the surface of the minor groove of the bases at
positions 7 and 8 and faces toward repressor (Fig.
1). It also forms a base pair hydrogen
bond with the carbonyl O2 on the opposing pyrimidine base. The third
base pair hydrogen bond contributed by the N2-NH2 group on
purines may decrease the affinity of repressor for DNA by resisting the
repressor-induced change in propeller twisting (3). Alternatively, the
N2-NH2 group on a purine at the center of the binding site
may decrease the affinity of repressor for DNA by sterically (3) or
electrostatically (11) hindering overwinding or narrowing of the minor
groove.
If the N2-NH2 group influences repressor binding by
resisting propeller twist, we anticipate that the affinity of repressor for DNA would decrease with an increase in the number of hydrogen bonds
between the bases at the center of the binding site. However, there is
no correlation between the number of base pair hydrogen bonds between
the bases at the center of the binding site and the affinity of
repressor for DNA (Fig. 2). Instead,
repressor has lower affinity for binding sites containing an
N2-NH2 group on the bases at positions 7 and 8 than it does
for sites that lack this functional group. For example, despite having
one more hydrogen bond between its central bases than does
T7·AP8, the T7·DAP8 site binds repressor with a higher
affinity than does T7·AP8. This effect is seen independent of the
number of base pair hydrogen bonds between the bases at the center of
the binding site. For example, the AP7·C8 site contains an
N2-NH2 group at position 7 and the central base pairs are
joined by a single H-bond, whereas the I7·C8 site lacks the
N2-NH2 group at position 7 and the I7·C8 base pair is
joined by two H-bonds. Nonetheless, repressor binds to the AP7·C8
with ~2500-fold lower affinity than it does to the I7·C8
site. Repressor does not closely approach the N2 position of the bases
at positions 7 and/or 8 (1, 3). Thus, the presence of the N2-exocyclic
NH2 group appears to interfere with high affinity binding
of repressor by opposing repressor-induced changes in DNA twist or
minor groove geometry.
Salt Concentration Dependence of the Affinity of 434 Repressor for
Position 7 and 8 Variant 434 Binding Sites--
To determine how the
N2-NH2 group influences the affinity of repressor for DNA,
we examined the effect of changing salt concentration on the affinity
of repressor for binding sites the do or do not contain an
N2-NH2 group on the purines at positions 7 and 8. We analyzed DNA sites that differ only in the absence or presence of the
N2-NH2 group at positions 7 (G7·C8 and I7·C8) or 8 (T7·A8 and T7·DAP8).
The affinities of 434 repressor for binding sites that do not have an
N2-NH2 group on the purine at positions 7 or 8 are
relatively unaffected by salt concentration (Fig.
3). In comparison, the affinities of
repressor for sites bearing an N2-NH2 group on the purines
at positions 7 and 8 are much more dependent on salt concentration (Fig. 3). The increased salt sensitivity of the affinities of repressor
for binding sites bearing an N2-NH2 group at positions 7 and 8 suggests that the stabilities of these repressor-DNA complexes are more dependent on ionic interactions between protein and DNA than
are repressor complexes with DNAs that lack this functional group on
the central bases.
Extrapolating the salt concentration dependence of affinity data to 1 M [KCl] allows determination of the salt-insensitive component of the binding affinity (12). This number, in part, reflects
the strength of specific protein-DNA contacts. This analysis shows that
the strength of the salt-insensitive protein-DNA contacts is decreased
between 102 and 104-fold in complexes between
repressor and the sites bearing the N2-NH2 group on the
bases at their centers, relative to those complexes that lack this
functional group on these bases (Fig. 3). Together, these data suggest
that the N2-NH2 group on bases at positions 7 and 8 destabilizes 434 repressor-DNA complexes by decreasing the intimacy of
the specific repressor-DNA contacts while increasing the reliance on
protein contacts to the DNA phosphate backbone.
Probes of 434 Repressor Binding Site Structure--
The salt
concentration dependence studies imply that the conformation of
repressor-DNA complexes vary with the absence or presence of an
N2-NH2 group on the purines at positions 7 or 8. We
examined whether the N2-NH2 group also affects the
conformation of unbound DNAs by CD spectroscopy. The CD spectrum of
each sequence is characteristic of a right-handed DNA double helix
(Fig. 4, A and B).
However, the relative intensities of the positive CD peak at ~275 nm
and the negative peak at ~250 nm are much lower for the binding sites
that lack the N2-NH2 group at the central positions than
for those that do have this group (Fig. 4, A and B, compare the spectrum of G7·C8 with I7·C8 and T7·A8
with T7·DAP8). The spectra of the heat-denatured DNAs are not
affected by the presence or absence of the N2-NH2 group
(data not shown). Thus, the base composition-dependent
changes in CD intensities are not due to variation in the extinction
coefficients between bases that do or do not contain the N2 group.
Hence, the differences in CD spectra between the sites that do or do
not contain the N2-NH2 group can be attributed to base
type-dependent changes in DNA conformation.
In general, lower CD intensity at 275 nm is indicative of higher
helical twist (13). Thus, the presence of an N2-NH2 group on the purine bases at positions 7 and 8 of the 434 binding site decreases the intrinsic twist of these sites, relative to the sites
that lack this feature. These findings are consistent with our previous
biochemical and NMR results showing that the DNA at the center of the
G7·C8 site is underwound and the minor groove in this region is wider
than that of the identical region of the T7·A8 site
(5).2 The 434 repressor
prefers to bind DNA sites that are overwound as opposed to those that
are underwound (6). Since I7·C8 and T7·A8 both exhibit the highest
affinity for repressor within their respective sequence contexts, the
CD results indicate that the presence or absence of the
N2-NH2 group at positions 7 or 8 may affect the DNA
affinity of 434 repressor by altering DNA twist.
The salt dependence data (Fig. 3) suggest that the structure of the
repressor-DNA complex also varies with the presence or absence of the
N2-NH2 group on the central bases. To explore this possibility, we used ·OH radical cleavage to examine the effect
of the N2-NH2 group on the conformation of the unbound and
bound DNAs. ·OH cleaves DNA by accessing the sugar phosphate
backbone through the minor groove and large N2-NH2
group-dependent changes in the geometry of the minor groove
of the binding sites would be reflected in changes in cleavage
intensity (10). Similarly, repressor-DNA complex formation will protect
the DNA from cleavage by making the minor groove less accessible to the
·OH radical (10). Differences in the ·OH cleavage
patterns of the 434 binding sites in the presence of saturating
concentrations of repressor will reflect differences in the
conformations of the repressor-DNA complexes.
In the absence of repressor, the intensity of ·OH radical
cleavage at each base in T7·A8 and T7·DAP8 does not differ
significantly, regardless of salt concentration (Fig.
5A; note that the apparent effect of salt on cleavage at position 2 is due to a salt effect on
electrophoretic mobility of this fragment). Similarly, the ·OH
radical cleavage patterns of I7·C8 and G7·C8 determined at 50 and
150 mM KCl in the absence of repressor are also
indistinguishable from each other (Figs. 5B and
6B). These finding suggests that neither salt nor the
presence of the N2-NH2 group at positions 7 and 8 in these
binding sites significantly affects the width of the minor groove
either at the site of substitution or globally throughout the binding
site DNA. This conclusion suggests that the N2-NH2
group-dependent twist differences in these DNAs seen by CD
spectroscopy (Fig. 4) are not detectable by ·OH radical or do
not translate into large alterations in the minor groove geometry of
the unbound DNA. Based on our NMR data (not shown), we favor the former
interpretation.
The cleavage pattern of the T7·A8-repressor complex is remarkably
different from that of the repressor-T7·DAP8 complex. This N2-NH2 group-dependent difference in cleavage
pattern is seen at both 50 and 150 mM KCl (Figs. 5 and
6). Marked differences in the cleavage
patterns of the G7·C8- and I7·C8-repressor complex are also seen,
regardless of salt concentration. Control experiments show that the
N2-NH2 group-dependent changes in ·OH
cleavage pattern are not due to differences in repressor occupancy of
the binding sites. Thus, the differences in ·OH cleavage
patterns must reflect N2-NH2 group-dependent
differences in the conformations of the repressor-DNA complexes.
N2-NH2 group-dependent changes in ·OH
cleavage pattern are seen throughout the binding site (Figs. 5 and 6),
but the most striking differences are seen at the center of the binding
site. Specifically, repressor more efficiently protects positions 9 and
10 of the sites that lack the N2-NH2 group at positions 7 and 8 (T7·A8 and I7·C8) than it does on sites bearing this
functional group at these positions (T7·DAP8 and G7·C8). For
example, at 50 mM KCl, repressor binding to T7·A8 reduces
the ·OH cleavage intensity at positions 9 and 10 8-fold and
4-fold, respectively (Fig. 6A). In contrast, under the same
conditions, repressor binding to T7·DAP8 reduces the cleavage
intensity at these positions by at most 2.5-fold. Even larger
differences in protection efficiency occur between the G7·C8 and
I7·C8 sites (Figs. 5B and 6B).
In complex with repressor, the minor groove of the bases at positions 9 and 10 faces the repressor (see Fig. 1). The N2-NH2 group
on positions 7 and 8 is located in this groove. Repressor makes no
direct contacts to the bases in this region of the binding site,
interacting only with the DNA backbone (1). The decreased efficiency of
protection of the DNA in this region in the repressor-T7·DAP8 and
G7·C8 complexes suggests that repressor is less intimately associated
with the DNA in this region of the binding site than in the repressor
complex with T7·A8 and I7·C8
The ·OH cleavage pattern at symmetrically related positions 1-4
and 11-14 also differs in the complexes between repressor and sites
that do or do not contain an N2-NH2 group on the central bases. For example, at 50 mM KCl repressor protects the
bases at positions 11-14 in T7·DAP8 binding to a greater extent than in the T7·A8 site (Fig. 6, A and B).
Differences are also seen at positions 1-4 (Fig. 6, A and
B). A similar set of N2-NH2
group-dependent differences in repressor-mediated
protection of the outer bases is seen between the G7·C8 and
I7·C8 sites (Fig. 6, C and D). Since repressor makes contacts to the DNA only from the major groove side of
the bases near positions 1-4 and 11-14 (Fig. 1) and since ·OH
gains access to the phosphate backbone through the minor groove, repressor binding must affect the ·OH reactivity of this region
indirectly. Thus, we assert that the differences in cleavage efficiency
at positions 1-4 and 11-14 between the repressor-DNA complexes that
do or do not contain an N2-NH2 group on the purine bases at
position 7 and 8 report an N2-NH2 group-mediated difference
in specific protein-DNA contacts. This finding is consistent with the
large difference in the strength of the salt-insensitive component of
the binding affinity between sites that do or do not contain the
N2-NH2 group on the purine bases at positions 7 and 8 (Fig.
3).
Our findings show that placing an N2-NH2 group on the
purine bases at positions 7 or 8 globally alters the conformation of the DNA and repressor-DNA complexes. Also, we know that repressor binds
with higher affinity to DNAs bearing central sequences that are
relatively flexible and/or overwound than it does to those that are
"stiffer" and/or relatively underwound (3, 5-7). How might the
N2-NH2 group exert its effects on the repressor-DNA complex
and/or DNA structure? Here, we consider two models. In a "twist/flex
model", the N2-NH2 group of guanine may directly impact
the DNA helical twist and/or twisting flexibility (5) by mechanical
occlusion (steric hindrance) (6). According to this idea, the
effects/interactions of the N2-NH2 are through space, are
not mediated by other species, and are therefore intrinsic to the DNA.
An alternative viewpoint advocates that the N2-NH2 alters
DNA conformation and flexibility via electrostatic
interactions of this group with its environment (11), influencing
distributions of protein-based or solution-derived cations within the
major and minor groove leading to sequence-dependent
variation in groove width, DNA twist, and axial bending (14). According
to this model, the N2-NH2 group interactions depend on
factors extrinsic to DNA.
Although extrinsic forces do play a role in 434 repressor-DNA
interactions,3 evidence
suggests that the effect of the N2-NH2 group on the central
bases on repressor-DNA complex stability is independent of the
electrostatic contributions of this group. First, the results of
mutagenesis studies (3) suggest that the partial positive charge on the
N2-NH2 group (11) is unimportant to the ability of
repressor to recognize the noncontacted base sequence at positions 7 and 8. Second, the presence of a partial positive charge in the minor
groove should cause DNA to overwind (15). Since our data indicate the
N2-NH2-group containing DNAs are underwound (16) with
respect to DNAs lacking this group (Fig. 4 and Ref. 5),2
we favor the idea that N2-NH2 groups sterically
oppose repressor-induced DNA distortions needed for stable
repressor-DNA complex formation. Structural (17-19), biochemical
(20-22), and molecular dynamic studies (23) are consistent with this idea.
Regardless of whether the steric or electrostatic "bulk" of the
N2-NH2 group on the central bases of the binding site is
responsible for its deleterious effects on repressor-DNA complex
stability, our results allow us to develop a picture of how the
N2-NH2 group influences strength and conformation of the
repressor-DNA complex. In agreement with our earlier results (5), the
CD data (Fig. 4) show that DNAs bearing the N2-NH2 group at
position 7 and 8 are underwound with respect to sites lacking this
group. Thus to form a complex, the repressor must overwind these DNAs.
The decreased twisting flexibility of the N2-NH2 group
containing DNAs contributes to the lower affinity of repressor for the
G7·C8 and T7·DAP8 sites (5, 23). In addition, the presence of an N2-NH2 group on the central bases of the binding site may
also inhibit the minor groove compression that normally accompanies repressor binding. Failure to compress the minor groove in this region
compromises the ability of repressor to establish specific contacts
with the bases at the outer edges of the binding site, at positions
1-4 and 11-14 (1, 24). Unfortunately our data cannot distinguish
which of these various N2-NH2 group-dependent changes causes repressor to bind the G7·C8 and T7·DAP8 sites with lower affinity. However, these findings suggest that in addition to its
effect on DNA flexibility, (5), the presence of the N2-NH2
group causes repressor to assume an unfavorable juxtaposition in its
complex with DNA, prohibiting repressor from making conformationally appropriate and thermodynamically strong interactions with the DNA bases.
Indirect readout of DNA sequence plays a role in determining the
stability and/or specificity of many protein-DNA complexes. Detailed
analysis of the sequence dependence of the structures of DNA both alone
and in complex with proteins shows that DNA has within it design
elements that allow it to assume a wide variety of structural variants.
Although this structural polymorphism is crucial to the biological
functions of DNA and protein-DNA complexes, precise knowledge of how
base sequence modulates DNA structure, and thereby indirect readout, is
lacking. The results reported here provide insights into how one of
these design elements, the N2-NH2 group of purines, may
influence the ability of a protein to recognize and/or coerce DNA into
a variety of non-canonical structures, a feature that is an absolute
prerequisite for many of the important DNA transactions that take place
in the genome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP in the presence of T4 polynucleotide
kinase (Invitrogen) for 30 min at 37 °C in a buffer containing 50 mM Tris, pH 8.0, and 10 mM MgCl2.
The labeled DNA was ethanol-precipitated and used in filter-binding
experiments as previously described (9). Values of the dissociation
constant (KD) were determined by nonlinear squares
fitting of the filter binding data using Prism 3.0 software (GraphPad
Software Inc.). Each dissociation constant was determined from at least
eight replicate measurements.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (63K):
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Fig. 1.
Schematic representation of the N-terminal
DNA binding domain of 434 repressor in complex with its binding site
(1). Indicated are the positions of the bases that are or are not
directly contacted by 434 repressor. Base numbering is from the 5' end
of the consensus 14 base pair-long binding site. The bases indicated by
X7 and Y8 correspond to positions substituted by noncanonical bases as
described in Fig. 2.
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Fig. 2.
Effect of base modifications on
KD of repressor for 434 binding sites.
Modified bases were substituted into positions 7 or 8 of a rotationally
symmetric 434 binding site sequence. Except for the substituted
positions (positions 7 or 8) the sequences of all the binding sites are
otherwise identical, as indicated in the sequence at the top. The
substitutions at positions 7 and 8 were made symmetrically, such that
the modified base appears at the indicated position on both strands.
The strands are numbered 1-14, starting from the 5' end of the
sequence. The dissociation constants shown were determined at 150 mM KCl as described under "Experimental
Procedures."
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[in a new window]
Fig. 3.
Salt concentration dependence of the affinity
of 434 repressor for binding sites bearing modified bases at their
central positions. Plotted is the log KEQ
versus log [KCl] for the position 8-substituted DNAs
(A), T7·A8 (
) and T7·DAP8 (
), and position
7-substituted DNAs, I7·C8 (
) and G7·C8 (
). The
lines represent linear least squares fits to these
data.
View larger version (22K):
[in a new window]
Fig. 4.
Circular dichroism spectra of 2 mM of DNAs bearing modified bases at their central
positions. Spectra were acquired at 50 mM KCl,
25 °C (see also "Experimental Procedures"). The spectra of
T7·A8 ( ) and T7·DAP8 (
) are shown in A. B displays the spectra of I7·C8 (
) and G7·C8
(
).
View larger version (60K):
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Fig. 5.
Hydroxyl radical footprinting analysis of the
DNA bearing N2-NH2-group changes at positions 7 or 8. Shown is a phosphorimage of the cleavage patterns of T7·A8 and
T7·DAP8 (A) and I7·C8 and G7·C8 (B)
obtained in the absence ( ) and presence (+) of 434 repressor at 50 and 150 mM KCl as indicated. The numbers denote the
sequence positions indicted in Fig. 2.
View larger version (15K):
[in a new window]
Fig. 6.
Relative intensity differences between
hydroxyl radical cleavage patterns of bound and unbound 434 repressor
binding sites. Shown is the fold difference in hydroxyl radical
cleavage intensity of unbound DNA relative to repressor-bound DNA for
DNAs bearing substitutions at position 8 (A and
B, white bars for T7·A8 and black
bars for T7·DAP8) and position 7 (C and D,
white bars for I7·C8 and black bars for
G7·C8) determined at 50 (A and C) and 150 (C and D) mM KCl.
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FOOTNOTES |
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* 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: Dept. of Biological
Sciences, University at Buffalo (SUNY), Cooke Hall, North Campus,
Buffalo, NY 14260-1300. Tel.: 716-645-2363, Ext. 158; Fax:
716-645-2975; E-mail: koudelka@acsu.buffalo.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212667200
2 G. B. Koudelka, J. Schwartz, and D. Gorenstein, unpublished results.
3 G. Koudelka and S. Mauro, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: CD, circular dichroism; DAP, diaminopurine; AP, amino purine.
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REFERENCES |
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1. | Aggarwal, A., Rodgers, D. W., Drottar, M., Ptashne, M., and Harrison, S. C. (1988) Science 242, 899-907[Medline] [Order article via Infotrieve] |
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