Eur. J. Biochem. 271, 3556–3566 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04292.x
Sequence selective binding of bis-daunorubicin WP631 to DNA
Keith R. Fox1, Richard Webster1, Robin J. Phelps1, Izabela Fokt2 and Waldemar Priebe2
1
School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, UK; 2The University of Texas MD
Anderson Cancer Center, Houston, TX, USA
We have used footprinting techniques on a wide range of
natural and synthetic footprinting substrates to examine the
sequence-selective interaction of the bis-daunorubicin antibiotic WP631 with DNA. The ligand produces clear
DNase I footprints that are very different from those seen
with other anthracycline antibiotics such as daunorubicin
and nogalamycin. Footprints are found in a diverse range of
sequences, many of which are rich in GT (AC) or GA (TC)
residues. As expected, the ligand binds well to the sequences
CGTACG and CGATCG, but clear footprints are also
found at hexanucleotide sequences such GCATGC and
GCTAGC. The various footprints do not contain any particular unique di-, tri- or tetranucleotide sequences, but are
frequently contain the sequence (G/C)(A/T)(A/T)(G/C). All
sequences with this composition are protected by the ligand,
though it can also bind to some sites that differ from this
consensus by one base pair.
A large number of ligands are known to bind to DNA, and
several of these are important therapeutic agents, particularly in the treatment of cancer. However most such agents
have little or no sequence selectivity and are therefore
extremely cytotoxic and affect all rapidly dividing cells. One
goal for cancer chemotherapy is therefore to produce
compounds that only interact with specific genes, or gene
products. The deciphering of many complete genomes gives
new impetus to the search for molecules that interfere with
the activity of individual genes. Examples of strategies
aimed at realizing this goal included the formation of
intermolecular triplex helices [1,2] and the pyrrole-imidazole
polyamines [3,4].
The interaction of many small molecules with DNA
has been well characterized and several of these have
limited sequence recognition properties. However, with
the exception of the polyamides [3,4] these compounds
only recognize between two and four base pairs. One
means of increasing the selectivity is to produce oligomers of known agents, thereby increasing the binding site
size, the selectivity and the strength of binding [5–7]. The
first examples of such agents included the bis-intercalating acridines. These generally bind more strongly than
simple mono-intercalators, though this rarely approaches
the theoretical limit of the square of the binding
constant, because of conformational and structural
restrictions imposed by the linkers between the two
intercalators. In addition, because the parent compounds
bind to almost all DNA sequences, the oligomers show
little or no sequence selectivity.
The anthracycline antibiotics are well known antitumour
agents [8–11] and, although they display a pleiotropic
mechanism of action, DNA is their primary cellular target.
The best characterized members of this group are daunorubicin (daunomycin) and doxorubicin (adriamycin). These
agents bind to DNA by intercalation, with the amino sugar
daunosamine positioned in the DNA minor groove. Several
crystal structures have been reported for the interaction of
these ligands with oligonucleotides, including CGTACG
[12,13], CGATCG [14,15] and TGTACA and TGATCA
[16]. They possess some sequence specificity and high
resolution footprinting has suggested that they bind best to
the sequences 5¢-(A/T)CG and 5¢-(A/T)GC [17–19].
There have been a number of attempts to produce bisintercalating daunorubicin derivatives, with increased affinity for DNA. In early studies these were linked through C13
and C14 as these are chemically accessible [20,21]. However
these positions are involved in DNA binding and the
modifications decreased the affinity of each monomer.
More recently dimers of daunorubicin have been produced
by linking between the C-4¢ or C-3¢ sugar positions [22,23].
These compounds were designed after examination of the
crystal structure of daunorubicin bound to CGTACG [13].
This structure contains two daunorubicin molecules which
are intercalated at the CpG steps with their amino sugars
facing each other at the centre of the complex, with the
3¢-amines separated by 6–7 Å. A p-xylyl linker was chosen
to link the two halves of the dimer generating WP631
(Fig. 1), linked at the 3¢-positions and WP652 (linked at the
modified 4¢-positions). These compounds show promising
biological activity and are significantly more cytotoxic than
doxorubicin against multi-drug resistant tumours [22,23].
WP631 has been shown to be an Sp1 site-specific drug [24],
an activator of nuclear factor-j B [25], and an inhibitor
of Tat transcription in HIV [26], as well as a general
antiproliferative agent.
Spectroscopic methods have been used to examine the
binding of WP631 to DNA [27]. Continuous variation
Correspondence to K. R. Fox, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX,
UK. Fax: +44 23 80594459; Tel.: +44 23 80594374;
E-mail: [email protected]
Abbreviations: DEPC, diethylpyrocarbonate.
(Received 4 June 2004, revised 14 July 2004, accepted 15 July 2004)
Keywords: WP631; anthracycline antibiotic; daunorubicin;
footprinting; sequence recognition.
FEBS 2004
WP631 sequence selectivity (Eur. J. Biochem. 271) 3557
These studies have demonstrated that WP631 binds
tightly to DNA by bisintercalation and assume that it
recognizes the sequence CG(T/A)(T/A)CG. However, there
have been no previous studies examining its sequence
binding preferences, though it has been demonstrated that
WP631 inhibits Sp1-activated transcription in vitro [24,30].
In this paper we examine the DNA sequence specificity of
WP631 using a range of footprinting techniques on several
different DNA fragments.
Materials and methods
Chemicals and enzymes
Oligonucleotides for preparing the various DNA fragments
were purchased from Oswel DNA service (Southampton,
UK). These were stored in water at )20 C, and diluted to
working concentrations immediately before use. Plasmid
pUC19 was purchased from Pharmacia. DNase I was
purchased from Sigma and stored at )20 C at a concentration of 7200 UÆmL)1. Restriction enzymes and reverse
transcriptase were purchased from Promega. WP631
(Fig. 1) was prepared as previously described [23].
DNA fragments
Fig. 1. Chemical structure of WP631. The carbon atoms at positions
13, 14, 3¢ and 4¢ are indicated in the upper part of the dimer.
analysis revealed up to six distinct binding modes to
herring sperm DNA. The tightest of these corresponded to
the interaction of one drug molecule with six base pairs
with an association constant of 3 · 1011 M)1 at 20 C.
High resolution melting studies showed that the ligand
bound preferentially to GC-rich DNA regions [27]. By
comparison with the crystal structures of daunorubicin
we would expect these ligands to bind to the sequence
CG(A/T)(A/T)CG. NMR [28] and crystal structures [29]
have been derived for the interaction of WP631 with
CGTACG and CGATCG, respectively, and as expected
show that the ligand binds by bisintercalation with each
chromophore inserted into the CpG steps, with four base
pairs sandwiched between them. In contrast, prolonged
incubation of WP652 with CGTACG resulted in precipitation, and the NMR structure was determined for this
ligand bound to TGTACA [28]. In this structure the ligand
is bound across the sequence PyGTPu, with only two base
pairs between the intercalated chromophores.
The sequences of the various fragments used in this work
are shown in the various differential plots (see below).
TyrT(43–59) is a 110 base pair fragment, which has been
widely used in previous footprinting studies [31]. This
labelled DNA fragment was obtained by cutting the
plasmid with EcoRI and AvaI and was labelled at the
3¢-end of the EcoRI site with [32P]dATP[aP] using reverse
transcriptase. Fragments MS1 and MS2 were designed to
contain all 136 possible tetranucleotide sequences [32].
These two fragments contain the same sequence in opposite
orientations, allowing visualization of footprints that are
located at either end. Fragments DMG60Y and DMG60R
contain oligopurine tracts which are interrupted with
different bases in the centre [33]. They contain the same
sequence in opposite orientations, they were radiolabelled
so as to visualize the purine-rich strand of DMG60R, and
pyrimidine-rich strand of DMG60Y. AG1 and GA1
contain the sequences A6G6.C6T6 and G6A6.T6C6 inserted
into the BamHI site of pUC18 [34]. Radiolabelling visualizes
the purine-rich strand of AG1, but the pyrimidine-containing strand of GA1. Fragments WPseq1 and WPseq2 were
obtained by cloning appropriate oligonucleotides into the
BamHI site of pUC18. The sequences were confirmed by
manual sequencing with a T7 sequencing kit (Amersham
Pharmacia). Fragment WPseq2 was found to contain a
dimer of the required insert. These fragments were obtained
by cutting the plasmids with HindIII and SacI and they were
labelled at the 3¢-end of the HindIII site with [32P]dATP[aP]
using reverse transcriptase. Radiolabelled DNA was separated from the remainder of the plasmid on 6–8% nondenaturing polyacrylamide gels. The bands containing the
radiolabelled DNA were excised and eluted into 10 mM
Tris/HCl pH 7.5, containing 0.1 mM EDTA. The DNA
was then precipitated with ethanol in the presence of 0.3 M
sodium acetate.
3558 K. R. Fox et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 2. DNase I, DEPC and KMnO4 footprints showing the interaction of WP631 with
tyrT(43–59). WP631 concentrations (lM) are
shown at the top of each gel lane; con corresponds to cleavage in the absence of added
ligand. Tracks labelled GA are markers
specific for purines.
DNase I footprinting
Radiolabelled DNA was dissolved in 10 mM Tris/HCl
pH 7.5 containing 0.1 mM EDTA, at about 10–20 c.p.s.
ÆlL)1 as determined on a hand-held Geiger counter. This
DNA solution (1.5 lL) was mixed with 1.5 lL of ligand
(final concentration 10 nM )10 lM), dissolved in 10 mM
Tris/HCl, pH 7.5, containing 10 mM NaCl. This mixture
was allowed to equilibrate for at least 30 min before
digesting with either DNase I or a hydroxyl radical
generating mixture as previously described [31]. DNase I
digestion was achieved by adding 2 lL enzyme (typically
0.01 UÆmL)1) dissolved in 20 mM NaCl, 2 mM MgCl2, and
2 mM MnCl2. The digestion was stopped after 1 min by
adding 5 lL of 80% formamide containing 10 mM EDTA,
10 mM NaOH and 0.1% (w/v) bromophenol blue.
Hydroxyl radical footprinting
Hydroxyl radical cleavage was performed by adding 6 lL of
a freshly prepared mixture containing 50 lM ferrous
ammonium sulfate, 100 lM EDTA, 2 mM ascorbic acid
and 0.05% hydrogen peroxide. The reaction was stopped
after 10 min by precipitating with ethanol. The DNA was
finally redissolved in 8 lL of 80% formamide containing
10 mM EDTA, 10 mM NaOH and 0.1% (w/v) bromophenol blue.
Reaction with diethylpyrocarbonate and potassium
permanganate
The reaction with these footprinting probes was performed
as previously described [29,31]. Radiolabelled DNA (3 lL)
was incubated with 3 lL WP631 diluted to appropriate
concentrations in 10 mM Tris/HCl containing 10 mM NaCl
and equilibrated for at least 30 min. For diethylpyrocarbonate (DEPC) modification, 5 lL of DEPC was added and
the reaction was stopped after 20 min by precipitating with
ethanol in the presence of 0.3 M sodium acetate. For
reaction with permanganate, 1 lL of 100 mM potassium
permanganate was added and the reaction stopped after
1 min by adding 2 lL of mercaptoethanol. The DNA was
then precipitated with ethanol in the presence of 0.3 M
sodium acetate. For both DEPC and permanganate the
dried DNA pellets were boiled in 10% (v/v) piperidine for
30 min, reduced to dryness in a Speedvac, and redissolved in
8 lL of 80% formamide containing 10 mM EDTA, 10 mM
NaOH and 0.1% (w/v) bromophenol blue.
FEBS 2004
WP631 sequence selectivity (Eur. J. Biochem. 271) 3559
Denaturing gel electrophoresis
Results
The products of footprinting reactions were resolved on
6–10% polyacrylamide gels (depending on the location of
the target site) containing 8 M urea. DNA samples were
boiled for 3 min immediately before loading onto the gels.
Polyacrylamide gels (40 cm long) were run at 1500 V for
2 h. These were then fixed in 10% acetic acid, transferred to
Whatmann 3M paper, dried under vacuum at 80 C and
exposed to a phosphorimager screen (Kodak) overnight.
Dry gels were exposed to a Kodak Phosphor Storage
Screen, which was scanned using a Molecular Dynamics
Storm 860 phosphorimager. The products of digestion were
assigned by comparison with Maxam–Gilbert marker lanes
specific for guanine and adenine.
Figure 2 shows footprinting gels for the interaction of
WP631 with the tyrT DNA fragment. This fragment has
been widely used for assessing the sequence-specific binding
of small molecules to DNA including the anthracycline
antibiotics daunorubicin and nogalamycin [17,19,35,36].
The first panel shows the results of DNase I footprinting,
from which it is clear that the ligand has affected the
cleavage pattern. At the highest concentrations of WP631
(2 and 3 lM), the ligand shows a general inhibition of
cleavage at most positions in the fragment. However specific
regions of protection are evident with ligand concentrations
between 0.2 and 1 lM. Examples of bands that are protected
by the ligand include positions 34, 41, 53 and 61. In contrast,
cleavage at positions 31–32 and 47–50 is enhanced in the
presence of the ligand. These results are presented as a
differential cleavage plot in the top panel of Fig. 3, showing
the intensity of each band in the drug-treated lanes
compared with that in the control. Examination of the
patterns does not reveal any obvious sequence preference,
though some of the clearest footprints are located in regions
containing both G and A residues. The enhancements are
located in oligo(dA) tracts, as often noted with intercalating
agents. These footprints are of variable lengths. The
footprints around positions 40, 63 and 80 cover about six
bases, as might be expected for a bis-intercalator. However,
below position 60 there are two smaller footprints of about
Differential cleavage plots
The intensity of bands in the drug-treated and control lanes
were prepared as previously described [31]. In the differential cleavage plots the intensity of each band in the drugtreated lane is divided by the intensity of the same band in
the drug-free control. These values are then normalized
according to the total intensity of the bands in each lane.
The values are then plotted against the DNA sequence on a
logarithmic scale. Values less than one correspond to
regions of protection by the ligand, while values greater
than one correspond to drug-induced enhanced cleavage.
Fig. 3. Differential cleavage plots showing the interaction of WP631 with tyrT(43–59), AG1 and GA1. The plots were calculated from the cleavage
patterns in the presence of 1 lM WP631 shown in Fig. 2 (tyrT) and Fig. 6 (AG1 and GA1). Only a part of each sequence is shown and is written
reading 5¢)3¢ from left to right; the right-hand end corresponds to the bottom of the gels. The ordinate, which is plotted on a logarithmic scale,
shows the intensity of each band in the drug-treated lanes relative to that in the control. Values less than one correspond to protection by the ligand,
while values above indicate enhanced cleavage. The black bars highlight the regions that are protected from cleavage. For tyrT the arrows indicate
the positions of WP631-induced cleavage by DEPC (grey arrows) and KMnO4 (black arrows).
3560 K. R. Fox et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 4. DNase I and KMnO4 footprints showing the interaction of WP631 with fragments MS1 and MS2. WP631 concentrations (lM) are shown at
the top of each gel lane; con corresponds to cleavage in the absence of added ligand. Tracks labelled GA are markers specific for purines. The
numbered black bars show the positions of DNase I footprints, while the asterisks indicate bands that become sensitive to reaction with KMnO4
in the presence of WP631.
three base pairs each. Similar short footprints are apparent
around positions 25 and 34. As DNase I overestimates
ligand binding site sizes by 3–4 base pairs it is very unusual
to observe footprints of this short size. It is possible that
these short footprints are not caused by steric interference
from drug molecules bound to the DNA minor groove,
instead they may reflect drug-induced changes in DNA
structure that render it less sensitive to cleavage. We
attempted to gain more accurate information about the
sequence specificity of WP631 by performing hydroxyl
radical footprinting experiments. However the ligand did
not affect hydroxyl radical cleavage at a concentration of
10 lM. Although this result is disappointing, some other
well-characterized sequence specific ligands also fail to
produce hydroxyl radical footprints [36,37].
Because of the difficulty in interpreting these patterns, we
examined the effect of WP631 on modification by DEPC
and potassium permanganate. These agents react with
exposed A and T residues, respectively, while duplex DNA
is generally unreactive [31]. Intercalating agents have
previously been shown to enhance the reactivity of bases
adjacent to their binding sites to these agents [38–40]. The
results are presented in the second and third panels of
Fig. 2. It can be seen that WP631 enhances the reactivity of
certain bases to each of these agents; these are indicated by
the arrows in Fig. 3. Bands that become hyper-reactive to
DEPC are located at positions 18, 32, 48, 67, 83 and 84. In
some instances these are located in regions of enhanced
DNase I cleavage (positions 32 and 48), while others are
adjacent to regions of DNase I protection (18, 67, 83, 84).
Enhanced reactivity to KMnO4 can be seen at positions 29,
33, 60, 68, 81, 86, 88 and 91.
These results show that WP631 produces distinct footprinting patterns, which are different to those produced by
daunorubicin and nogalamycin [17,19,35,36]. The ligand
must therefore possess some sequence selectivity, though no
consensus binding sites can be deduced from these patterns.
We have therefore examined the interaction of this ligand
with a range of DNA fragments, in order to elucidate the
characteristics of the preferred binding sites.
FEBS 2004
WP631 sequence selectivity (Eur. J. Biochem. 271) 3561
Fig. 5. Differential cleavage plots showing the interaction of WP631 with MS1, MS2 and DMG60. The plots were calculated from the cleavage
patterns in the presence of 1 lM WP631 shown in Fig. 4 (MS1 and MS2) and Fig. 6 (DMG60Y). Only a part of each sequence is shown and is
written reading 5¢)3¢ from left to right; the right-hand end corresponds to the bottom of the gels. The ordinate, which is plotted on a logarithmic
scale, shows the intensity of each band in the drug-treated lanes relative to that in the control. Values of less than one correspond to protection by
the ligand, while values above indicate enhanced cleavage. The black bars highlight the regions that are protected from cleavage. For MS1 and MS2
the arrows indicate the positions of WP631-induced cleavage by DEPC (grey arrows) and KMnO4 (black arrows).
Fragments MS1 and MS2 were designed so as to contain
all 136 tetranucleotide sequences [32]. They contain identical
sequences, but are cloned in opposite orientations thereby
simplifying analysis of bands at the ends of the fragments.
Footprinting experiments with these fragments are presented in Fig. 4 and differential cleavage plots derived from
these data are shown in Fig. 5. Again it is clear that WP631
has altered the cleavage patterns, producing footprints that
are highlighted by the bars in Figs 4 and 5. Several of these
footprints are located in regions which are rich in GA (TC)
or GT (AC) residues, for example sites 1 (TCATCTC),
2 (GGTGG), 4 (GAAGAG), 7(ATGTGT), and 8
(GTTGG). A long footprint is also evident on MS2 (site
10) corresponding to a purine-rich tract. These footprints
are accompanied by enhancements in reactivity to KMnO4
and DEPC as indicated in Figs 4 and 5.
As many of the footprints on MS1 and MS2 are located
in tracts of GA-residues we examined the interaction with
other fragments containing similar sites, some of which were
prepared for work with triplex-forming oligonucleotides.
Fragments GA1 and AG1 contain tracts of G6A6.T6C6 and
A6G6.C6T6, respectively [34]. DNase I footprinting patterns
for WP631 with these fragments are shown in Fig. 6 and
differential cleavage plots derived from these are presented
in Fig. 3. As these oligopurine tracts were both cloned into
the polylinker site of pUC18 the sequences surrounding the
inserts are common to both fragments and show similar
cleavage patterns in the presence of the ligand. For both
fragments there is a large footprint below the insert,
corresponding to the sequence TCCTCT. Similarly cleavage
is attenuated above the inserts in vicinity of the sequence
GGATC. However the ligand has very different effects on
cleavage of the two inserts. WP631 protects from DNase I
cleavage at the centre of AG1, but causes enhanced cleavage
at the centre of GA1. It therefore appears that AnGn is a
much better binding site than GnAn.
Fragment DMG60 also contains oligopurine tracts that
are interrupted by isolated thymine residues [33]. DNase I
digestion patterns for the pyrimidine-rich strand of this
fragment in the presence of WP631 are shown in Fig. 6 and
differential cleavage plots for both strands are shown in the
bottom panel of Fig. 5. Two clear footprints can be seen on
this fragment (labelled sites 1 and 2), as well as other regions
of protection at the top and bottom of the gel, which are in
the remainder of the polylinker. A short region of protection
is also evident around the lowest purine residue (arrowed).
The strong footprints correspond to sequences TTCTTC
(site 1) and TTTCTTT (site 2). Although these both contain
the sequence TTCTT, this alone cannot constitute the
preferred ligand binding site as the same pentanucleotide is
present in other positions which are not protected. These
will be considered further in the Discussion.
As several of the footprints identified above are located in
GA (CT) or GT (AC) tracts we prepared a new fragment
(WPseq2) containing five variations on the hexanucleotide
sequence SWSWWS (S ¼ G or C, W ¼ A or T), in which
the different sites are separated by CC (GG). The results of
DNase I footprinting experiments with this fragment are
3562 K. R. Fox et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 6. DNase I footprints showing the interaction of WP631 with fragments AG1, GA1
and DMG60Y. WP631 concentrations (lM)
are shown at the top of each gel lane; con
corresponds to cleavage in the absence of
added ligand. Tracks labelled GA are markers specific for purines. The numbered black
bars show the positions of DNase I footprints
with DMG60Y.
shown in Fig. 7. It can be seen that there are footprints at all
the potential sites, which are most clearly seen in the
differential cleavage plot. The strongest sites are at
GTGTTG and CTTCTC. There is little or no protection
in the junctions between the various sites and there is
enhanced cleavage between GTGGTG and CCACAC.
These regions of protection are located towards the 3¢-end
of each target site as normally observed with DNase I
footprinting, as this enzyme cuts across the width of the
DNA minor groove.
Daunorubicin is thought to bind best to sequences of the
type 5¢-(A/T)CG and 5¢-(A/T)GC [17–19] and previous
NMR and crystallographic studies with bis-daunorubicins
have investigated their interaction with CGTACG and
TGTACA [28,29]. None of these sequences are represented
in any of the footprinting substrates mentioned above. We
therefore prepared a novel fragment (WPseq1) containing
the sites CGATCG, CGTACG, GCATGC, GCTAGC and
TGTACA each separated by the sequence AATT to which
the drug is not expected to bind. The results of footprinting
experiments with this fragment are presented in Fig. 8. The
DNase I cleavage patterns show footprints at each of these
sites, some of which persist to between 0.1 and 0.2 lM. The
positions of these sites are confirmed in the differential
cleavage plot shown in Fig. 8(B). Although DNase I
footprinting cannot usually be used to determine ligand
binding sites to single base resolution, some interesting
features of WP631 binding can be deduced by comparing
the protection at each of these potential sites. The central
portions of the differential cleavage plots are four bases
long for GCATGC, CGATCG and TGTACA and each
begin at the second base. These footprints are symmetrically
located around the centre of each hexanucleotide target,
whereas DNase I footprints are usually staggered towards
the 3¢-end. In contrast the footprint at GCTAGC is longer
and begins one base before the start of this hexanucleotide;
it is therefore staggered towards the 5¢-end of the hexanucleotide site. The footprint at CGTACG appears to
consist of two smaller regions and there is little protection at
the central adenine. It should be remembered that all these
sites are symmetrical (palindromic) sequences. If the ligand
binds to one side of the site then a second identical site will
be present in the other half of the hexanucleotide (i.e. if it
binds to GCTAGC by recognizing GCT, then a second
identical binding site must be present in the other half of the
sequence at AGC). Although two ligand molecules will not
be able to bind simultaneously, the average of the two
equivalent binding sites would be a larger footprint, which is
not what we observe. It therefore seems most likely that a
single ligand molecule is bound across the centre of each
site. These differences between these sites are also evident in
the patterns of DEPC enhancement, which are indicated
by the arrows in Fig. 8. There is enhanced DEPC reactivity
at the first adenine after the hexanucleotide site (AATT) for
FEBS 2004
WP631 sequence selectivity (Eur. J. Biochem. 271) 3563
Fig. 7. Interaction of WP631 with fragment WPseq1. (A) DNase I footprints showing the interaction of WP631 with fragment WPseq2. WP631
concentrations (lM) are shown at the top of each gel lane; con corresponds to cleavage in the absence of added ligand. Tracks labelled GA are
markers specific for purines. The potential hexanucleotide binding sequences are indicated alongside the gel. (B) Differential cleavage plot showing
the interaction of WP631 with WPseq2. The plots were calculated from the cleavage patterns in the presence of 1 lM WP631 shown in Fig. 7A.
Only a part of each sequence is shown and is written reading 5¢)3¢ from left to right; the right-hand end corresponds to the bottom of the gel. The
ordinate, which is plotted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control. Values less
than one correspond to protection by the ligand, while values above indicate enhanced cleavage. The vertical lines divide the fragment into the
various hexanucleotide repeats.
GCATGC and GCTAGC, while this is at the second
adenine (AATT) for CGATCG and TGTACA. There is no
enhancement in reactivity to DEPC after CGTACG. These
subtle differences suggest that WP631 does not have exactly
the same mode of binding at each of these sites.
Discussion
The footprinting results presented in this paper demonstrate
that WP631 binds to DNA in a sequence selective fashion
and that its preferred binding sites are different from those
of daunorubicin and nogalamycin. By comparison with
daunorubicin it was expected that WP631 should bind best
to sequences such as CGATCG and CGTACG, which have
been used in X-ray and NMR structural studies with this
ligand [28,29]. The experiments with fragment WPseq2
confirm that WP631 does indeed bind to this site at
concentrations as low as 0.2 lM, but experiments with this
and other fragments show that it also binds equally well to
other sequences.
Another difference between these patterns and those
produced by daunorubicin is their temperature dependence.
Previous studies with daunorubicin [17–19] only detected
DNase I footprints at low temperature (4 C) presumably
as this slows the dissociation of the ligand from DNA; no
footprints were observed at 20 C. In contrast WP631
produces clear footprints at 20 C which are still apparent at
37 C. In this case we observe no WP631 footprints at 4 C.
This could be because the DNA becomes too rigid to permit
bis-intercalation, or because self-stacking of the ligand is
favoured at lower temperatures.
Mode of binding
Although the present work does not directly concern the
mode of binding of WP631, this will influence the
interpretation of the footprinting patterns. Previous structural work has demonstrated that WP631 binds in the minor
groove of CGTACG with four base pairs sandwiched
between the intercalating chromophores. In contrast, the
related compound WP652, in which the dimer is connected
via C4¢, binds to the YGTR steps in TGTACA, sandwiching only two base pairs between the chromophores. The
precise orientation of the xylyl group of WP631 is also
different in the two structures in which it is either
perpendicular or parallel to the walls of the minor groove.
3564 K. R. Fox et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 8. Interaction of WP631 with fragment WPseq2. (A) DNase I and DEPC footprints. WP631 concentrations (lM) are shown at the top of each
gel lane; con corresponds to cleavage in the absence of added ligand. Tracks labelled GA are markers specific for purines. The potential
hexanucleotide binding sequences are indicated alongside the gel. (B) Differential cleavage plot showing the interaction of WP631 with WPseq1. The
plot was calculated from the cleavage patterns in the presence of 0.2 lM WP631 shown in Fig. 8A. Only a part of each sequence is shown and is
written reading 5¢)3¢ from left to right; the right-hand end corresponds to the bottom of the gel. The ordinate, which is plotted on a logarithmic
scale, shows the intensity of each band in the drug-treated lanes relative to that in the control. Values of less than one correspond to protection by
the ligand, while values above one indicate enhanced cleavage. The arrows indicate the positions of WP631-induced cleavage by DEPC.
These different structures suggest that WP631 may bind to
different sequences in different modes, sandwiching between
two and four base pairs between the chromophores. These
different modes will depend on the local DNA structure and
flexibility as well as any contacts between the ligand and its
binding site. A further complication is the possibility that
WP631 might bind to some sequences by mono-intercalation, leaving the second chromophore in free solution or
stacked within the groove. The possibility of additional
sequence-specific groove binding may further complicate
the footprinting pattern. The coexistence of different
binding modes is suggested by the footprinting data
presented in this paper. Some binding sites are six to eight
base pairs long, as expected for a ligand that spans six base
pairs, while others are much shorter, and appear to cover
only three bases. These results are consistent with a recent
study suggesting that WP631 can bind in two different
modes with stoichiometries of 6 : 1 and 3 : 1 base pairs per
drug [41].
Sequence selectivity
The results with these DNA fragments show that
WP631binds to DNA in a sequence selective fashion, as
specific footprints are generated at moderate ligand concentrations (about 0.3 lM). At high concentrations (3 lM
and above) the ligand is able to bind to most sites, as shown
by the general inhibition of DNase I cleavage. Examination
of the footprints does not reveal the presence of any
particular di- or tri-nucleotide step within the binding sites,
though many of the protected regions are GA or GT-rich in
one strand, and there are no footprints in GC- or AT-rich
sequences. The results with MS1 and MS2, which contain
every possible tetranucleotide combination, demonstrate
that WP631 does not bind to a unique tetranucleotide,
though we cannot exclude the possibility that it binds
especially well to a unique hexanucleotide which is not
represented in these fragments. Several of the footprints are
found in oligopurine-oligopyrimidine sequences, especially
those seen with fragment DMG60. In the published crystal
[29] and NMR structures [28], WP631 is bound to the
sequences CGATCG and CGTACG, with the chromophores intercalated between each of the CpG steps. This
sequence is present in fragment WPseq2 and is indeed part
of a clear DNase I footprint, though several other sequences
produce equally good footprints on this fragment. It is
therefore clear that WP631 can bind to many sites with
the general sequence (G/C)(G/C)(A/T)(A/T)(G/C)(G/C).
A footprint is also evident in this fragment at the sequence
TGTACA, which was suggested as one of the potential
binding sites for WP652 [29]. We therefore examined the
footprinting results on all the fragments for degenerate
sequences that might form the preferred binding sites.
We find that footprints are often found around the sequence
(G/C)(A/T)(A/T)(G/C), and that there are no occasions
when this is not part of a drug binding site. For example, on
FEBS 2004
MS1 the footprints are at site 1 (CATC), site 3 (GTAC) and
site 4 (GAAG), while on MS2 they are seen at site 7
(CATG), site 8 (GTTG), site 9 (CTTG and GATC). In
addition the weaker regions of protection between sites 8
and 9 contain the sequences CTAC and CTAG. This
consensus sequence is also found on the tyrT fragment at
positions 25 (CATC), 38 (GTTG), 43 (GAAC) and 57
(GAAG) each of which corresponds to a region that is
protected by the ligand. The sequences GATC and CTGA
are also found in the polylinker regions of pAG1 and
pGA1, and at site 2 in DMG60 (CTTC). We therefore
suggest that WP631 binds well to the sequence (G/C)(A/
T)(A/T)(G/C). However, this sequence cannot be the only
good ligand binding site. For example, the footprint at the
centre of pAG1 contains the sequence AGGG (in contrast
to GGGA, which does not produce a footprint with pAG2).
Moreover sites 2, 5 and 6 on MS1 are found around the
sequences GGTG (or GTGG) (site 2), TTAG (site 5) and
GTATAG (site 6). The footprint at site 2 of DMG60 also
does not fit this pattern, and at this position it seems likely
that the ligand is able to bind to the two adjacent sites
CTTT. It therefore appears that the ligand can also bind
well to some sites in which one or more base does not match
the predicted pattern.
It should be noted that, although these results show the
presence of specific binding sites for WP631, these are
typically only evident at concentrations of 0.3 lM and
above. In contrast, previous studies have suggested that
WP631 binds with an association constant of 3 · 1011 M)1
[27], from which we would expect footprints to persist to
much lower (subnanomolar) concentrations. A number of
factors may contribute to this difference. First, it is possible
that the preferred binding site is not represented in the
footprinting substrates that we have used. We consider that
this is unlikely as the high affinity binding sites previously
reported were abundant with mixed sequence DNAs.
Second, in our footprinting experiments the substrate
DNA concentration is about 10 nM, and we will not be
able to detect stronger binding sites. Third, it is known that
WP631 strongly self-associates and the total ligand concentration may overestimate the concentration of the free
momomer.
Acknowledgements
This work was supported by grants from the Cancer Research UK, the
Association for International Cancer Research and The Welch
Foundation, Houston, Texas, USA.
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