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INTRODUCTION |
The long term survival for testicular cancer patients treated with
cis-diamminedichloroplatinum(II)
(cisplatin),1 exceeds 90%
(1). If the detailed mechanism of action of this anticancer drug could
be determined, analogs might be designed to broaden its organotropic
profile. It is generally accepted that the biological target of
cisplatin is DNA, the major adducts formed being 1,2-intrastrand d(GpG)
and d(ApG) cross-links involving platinum binding to the N-7 atoms of
the purine bases (2). Formation of these adducts unwinds the DNA and
bends it toward the major groove (3-7), blocking transcription and
replication. The ability to distort DNA structure cannot fully explain
the anticancer activity because the geometric isomer of
cisplatin, trans-diamminedichloroplatinum(II), which also
forms DNA adducts that modify the structure and block transcription and
replication, is clinically ineffective. Efforts have therefore focused
on identifying other cellular constituents that might interact
specifically with cisplatin-DNA adducts to effect the antineoplastic response.
By screening human and yeast cDNA expression libraries, genes were
discovered for proteins that bind specifically to cisplatin-modified DNA (8, 9). These proteins, human structure-specific recognition protein (hSSRP1) and intrastrand cross-link recognition protein (Ixr),
both contain 80-amino acid peptides homologous to the DNA-binding domains of the chromosomal protein HMG1. HMG1 is the prototypical member of a family of proteins that contain such DNA-binding motifs. Most HMG-domain proteins bind specifically to the major cisplatin-DNA adducts, increasing the bend angle by up to 60°, as determined through gel mobility shift assays (10-13). HMG domain proteins shield
cisplatin-DNA adducts from excision repair both in yeast cells and in
human cell extracts, suggesting that they may play a role in the
mechanism of action of cisplatin (11, 14, 15). SSRP1 has recently been
associated with a factor involved in transcription elongation,2 offering further
clues for how these proteins might be involved in the antitumor activity.
In the present work, fluorescence resonance energy transfer (FRET) was
developed as a probe for the further characterization of HMG-domain
protein binding to cisplatin-modified DNA. This technique is based on
the coupling of transition dipoles between two fluorescent dyes, a
donor and an acceptor, attached to a macromolecule. The efficiency of
the energy transfer between donor and acceptor depends on the distance
between them (16). As the distance decreases, the efficiency of the
energy transfer should increase. The FRET technique responds to small
changes in distance in the range of 10-75 Å (16) and can reveal
increased bending caused by an HMG-domain protein binding to
cisplatin-modified DNA. In addition, FRET has the potential to
facilitate kinetic studies of the interaction.
FRET has been used extensively to examine different types of nucleic
acid structures (17-23). It has also been applied to study the
structure of protein-DNA complexes (24-27). Recently, FRET techniques
were used to investigate bending within a 30-bp oligonucleotide that
accompanies the binding of the HMG-domain protein Chironomus HMG1 (26). The FRET distance measurements determined for the Chironomus HMG1-DNA complex enabled a bend angle of 150°
to be estimated for the DNA in that complex. Experiments undertaken here similarly examine the bend angle induced when an HMG domain protein binds to cisplatin-modified DNA. A 20-bp oligonucleotide probe
modified with fluorescein as the donor and rhodamine as the acceptor
was synthesized having a single cisplatin cross-link. Studies of the
binding of HMG1 domain B to this FRET probe revealed the increased
bending that accompanied protein-DNA complex formation, demonstrating
the viability of this approach.
In addition to examining the structural change that accompanies HMG1
domain B binding, FRET was employed to monitor the kinetics of the
interaction by using stopped-flow techniques. Stopped-flow fluorescence
spectroscopic experiments have afforded information about the rates of
binding for several other protein-DNA complexes (25, 28-31). In these
studies, changes in either the intrinsic protein fluorescence or in a
DNA probe containing a fluorescent label, resulting from protein-DNA
complex formation, were monitored over time. The kinetics of yeast
TATA-binding protein interacting with promoter DNA were revealed by
FRET (25). As in the present work, these experiments used a
fluorescein- and rhodamine-labeled DNA probe to follow protein-DNA
complex formation. Here we provide the first quantitative information
about the rates at which an HMG-domain protein binds to and dissociates
from cisplatin-modified DNA.
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EXPERIMENTAL PROCEDURES |
Materials and Methods--
Distilled, deionized water from a
Milli-Q system (Millipore) was used to prepare aqueous solutions.
Atomic absorption spectrometry was performed on a Varian AA1475
instrument with a GTA95 graphite furnace. High performance liquid
chromatography (HPLC) was carried out by using a Waters 600E system
controller equipped with either a Waters 484 or 486 detector.
Expression and Purification of Recombinant Rat HMG1 Domain
B--
This protein was obtained as described (13) with the following
modifications. Escherichia coli cells were grown in LB media and lysed by sonication. After passing through the S-Sepharose column,
the protein was purified by using an FPLC Superdex 75 size exclusion
column (Amersham Pharmacia Biotech). The eluent for the Superdex 75 column was phosphate-buffered saline (1 mM KH2PO4, 10 mM
Na2HPO4, 0.137 M NaCl, and 2.7 mM KCl, pH 7.4) with a flow rate of 0.5 ml/min.
Synthesis and Purification of Rhodamine- and Fluorescein-labeled
Oligonucleotide Probes--
The 20-bp oligonucleotide probes with
N-TFA C6 Amino Modifier (Cruachem) attached to the 5' end were
synthesized on a Cruachem PS250 DNA synthesizer by using standard
cyanoethylphosphoramidite chemistry. The crude deprotected
oligonucleotides were purified by size exclusion chromatography on a
G-25 Sephadex (Amersham Pharmacia Biotech) column using distilled,
deionized water as the eluent. The top strand was modified with 5- and
6-carboxytetramethylrhodamine, succinimidyl ester (Molecular Probes),
and the bottom strand was modified with 5- and 6-carboxyfluorescein,
succinimidyl ester (Molecular Probes). The composition of the reaction
mixture was 200 µM oligonucleotide, 5 mM
rhodamine or fluorescein, respectively, and 0.1 M sodium
carbonate buffer, pH 9.0. The reactions were stirred at room
temperature for approximately 72 h. When the reactions were
complete, excess unreacted dye was removed by using Centriplus 3 concentrators (Amicon) followed by dialysis (Mr
cut-off 1000, Spectra/Por).
The FRET probes were purified by reverse phase HPLC on a Vydac C4
preparatory column (22 × 250 mm, 10-µm particle size) using a
flow rate of 10 ml/min. Buffer A was 0.1 M
NH4OAc, and Buffer B was 0.5 M
NH4OAc in 50% CH3CN. The gradient used was
90% Buffer A to 60% Buffer A over 30 min, followed by 60% Buffer A
to 50% Buffer A over 10 min. Because the rhodamine and fluorescein
dyes employed in the syntheses were a mixture of the 5- and
6-succinimidyl ester isomers, two FRET probes were observed for each
synthesis in the HPLC trace. The 5-isomer was the dominant product,
eluting with a greater retention time for oligonucleotides labeled with both rhodamine (~25 min) and fluorescein (~20 min), and therefore was used in the FRET experiments. Electrospray mass spectrometry was
used to characterize the rhodamine-labeled top strand and fluorescein-labeled bottom strand. The molecular mass determined for
the rhodamine-labeled top strand with one sodium ion was 6543.45 Da
(calculated mass, 6543.03 Da), and the molecular mass determined for
the fluorescein-labeled bottom strand with one sodium ion was 6819.45 Da (calculated mass, 6819.03 Da).
Reaction of Cisplatin with the Rhodamine Probe--
The purified
rhodamine probe was lyophilized, dissolved in distilled, deionized
water, and passed through a Dowex 50 × 8-200 (Aldrich) ion
exchange column to exchange the ammonium ions for sodium ions. An
activated cisplatin solution was made by reaction with 1.97 mol eq of
AgNO3 (Aldrich) in distilled, deionized water on a shaker
at room temperature overnight in the dark followed by centrifugation to
remove the AgCl precipitate. The rhodamine-modified probe was
platinated by addition of the activated cisplatin solution (2 eq) to a
100 µM solution of the oligonucleotide. The reaction mixture was incubated at 37 °C for ~3 h. The product was purified by HPLC, using a Dionex Nucleopac PA-100 (9 × 250 mm) preparatory column at a flow rate of 4 ml/min. It was then eluted with a gradient of 75% Buffer A (0.025 M NH4OAc in 10%
CH3CN) to 50% Buffer A/Buffer B (0.025 M
NH4OAc, 1 M NaCl in 10% CH3CN)
over 30 min. The collected peak was dialyzed (Mr
cut-off 1000, Spectra/Por) against distilled, deionized water for
48 h. Platinum atomic absorption spectroscopy revealed there to be
one platinum atom per oligonucleotide strand.
Synthesis and Purification of Unlabeled Oligonucleotide
Probes--
A portion of the 20-bp top strand with the C6 amino
modifier described above was purified by HPLC on a Dionex Nucleopac
PA-100 (9 × 250 mm) preparatory column. The flow rate was 4 ml/min, and the gradient and buffers were as described above for the
rhodamine probe. The collected peak was dialyzed as above. This top
strand was used to make the unplatinated, fluorescein-labeled control duplex probe (Fl-ds). The unlabeled cisplatin-modified top strand was
made as described previously (32) and used to make the platinated, fluorescein-labeled control duplex probe (Pt/Fl-ds).
Purification of the Fluorescent Duplex Probes--
Fig.
1 shows a schematic drawing of the four
different oligonucleotide duplex probes and their abbreviations. The
fluorescein-labeled bottom strand was combined with unlabeled top
strand, with unlabeled, cisplatin-modified top strand, with
rhodamine-labeled top strand, or rhodamine-labeled, cisplatin-modified
top strand to make the four different FRET probes. Equimolar amounts of
the top and bottom strands were combined in a solution containing 10 mM Tris, pH 7.0, 50 mM NaCl, and 10 mM MgCl2. The solution was heated to 90 °C
and slowly cooled to 4 °C over several hours. The duplex DNA was
purified by ion exchange HPLC to remove excess single-stranded material. A Dionex Nucleopac PA-100 (9 × 250 mm) preparatory
column was used at a flow rate of 4 ml/min and a gradient of 70%
Buffer A to 45% Buffer A over 30 min, followed by 45% Buffer A to 0% Buffer A over the next 10 min. Under these conditions, the duplexes eluted at ~35 min. The collected samples were dialyzed against distilled, deionized water for 48 h as above, and the resulting solutions were lyophilized to dryness. The duplex samples were redissolved in 10 mM Tris, pH 7.0, 50 mM NaCl,
and 10 mM MgCl2. The solutions were heated to
90 °C and slowly cooled to 4 °C over several hours. A Dionex
Nucleopac PA-100 (4 × 250 mm) column was used to check the purity
of the final duplex materials by analytical ion exchange HPLC.

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Fig. 1.
Description of the oligonucleotides used in
FRET studies. Fl is fluorescein, and Rh is
rhodamine. The structures of the succinimidyl ester forms of the dyes
used to label the oligonucleotides are depicted. Fl-ds is
the fluorescein-labeled probe. Pt/Fl-ds is the platinated
fluorescein-labeled probe. Rh/Fl-ds is the rhodamine- and
fluorescein-labeled probe. Pt/Rh/Fl-ds is the platinated
rhodamine- and fluorescein-labeled probe. The 1,2-intrastrand d(GpG)
cisplatin adduct is denoted by asterisks.
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Fluorescence Titration Experiments with HMG1 Domain B--
These
experiments were performed with a Hitachi F-3010 spectrofluorimeter at
25 °C. Emission spectra were recorded of both the unmodified and
cisplatin-modified duplex probes labeled with both fluorescein and
rhodamine in 10 mM HEPES, pH 7.0, and 200 mM
NaCl solution in the presence of increasing concentrations of HMG1
domain B. The excitation wavelengths used were 480 and 550 nm, and
fluorescence emission spectra were observed at 490-610 and 560-610
nm, respectively. Emission spectra obtained from exciting at 550 nm
(rhodamine fluorescence only) were used to correct the data for
dilution and photobleaching effects. The corrected emission spectral
data at 520 nm were used to determine the dissociation constant,
Kd.
Kd values were determined by plotting the change in
fluorescence intensity at 520 nm,
F, against the total
protein concentration, [Pt]. The data were fit
to the expression described in Equation 1, where
[Dt] is the total oligonucleotide
concentration and c is a constant that relates fluorescence
intensity (I) to concentration. The average value of
c was determined to be 1.8 ± 0.5 × 10
9 M I
1. This equation is
derived directly from the equilibrium mass action expression with no
assumptions. The value of [Dt] was set to be
the concentration of DNA used in each titration experiment.
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(Eq. 1)
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Fluorescence Lifetime FRET Experiments--
Time-correlated,
single photon counting methods were employed to determine the
fluorescence lifetime of the fluorescein in the oligonucleotide duplex
probes. Light excitation was accomplished with a synchronously pumped,
mode-locked, cavity-dumped picosecond dye laser system. The PUMP laser
was a mode-locked Coherent Antares 76-s Nd:YAG
(neodymium:yttrium-aluminum-garnet) system, which produced 22 W of
1.06-µm radiation in approximately 80-ps pulses at a repetition rate
of 76.0 MHz. External frequency doubling and tripling units (CSK
Optronics) were utilized to generate 1.6 W of 355-nm light, which
pumped the laser dye coumarin 2 in the home-built system consisting of
an extended-cavity Coherent 590 dye laser equipped with a Coherent 7220 cavity dumper. Approximately 15 mW of 450 nm light was generated at a
repetition rate of 7.6 MHz, with pulse width of ~5 ps.
The sample was contained in a 1-ml fluorescence cuvette (10 × 10 mm), surrounded by an ice bath to ensure constant temperature, and
irradiated within a light-tight box. The fluorescence was detected at
right angles to the laser beam; no collection optics were employed, but
the size of the active area on the microchannel plate photomultiplier
tube (MCP-PMT; 2.0 inches in diameter) and its proximity to the sample
(6 inches) ensured nearly f/3 collection efficiency. A
combination of colored glass filters (Schott) was employed to act as a
notch filter and block from the detector both scattered laser light and
emission from the rhodamine on the fluorescent duplexes. This filter
combination passed only a narrow band of wavelengths around 500 nm (30 nm, full width at half-maximum).
Fluorescence was detected with a two-stage MCP-PMT (Hamamatsu R1564u)
having a rise time of 70 ps. The MCP-PMT output was directed through
two gain 10, 2.0-GHz preamplifiers (Sonoma Instrument) and then into a
constant fraction discriminator (Tennelec TC454). To take advantage of
the high repetition rate of the laser, the time-to-amplitude converter
(Canberra 2145) was utilized in "reverse" mode, such that pulses
from the MCP-PMT provided the "start" pulse, and a time-delayed
photodiode (Antel Optronics, ARX-SP, 210-ps rise time) pulse provided
the "stop" pulse. The ratio of the photon detection rate to the
laser repetition rate was at all times less than 1%, and usually below
0.001. The output of the time-to-amplitude converter was temporarily
stored in a multichannel analyzer (Nucleus, Inc., Oak Ridge, TN) and
processed on a personal computer. Typical signal integration time was 5 min for one decay measurement.
The fluorescence lifetime of the fluorescein dye was measured for 16 different samples by using the apparatus described above. The
concentration of all DNA probes was 40 nM duplex in 10 mM HEPES, pH 7.0, and 200 mM NaCl. HMG1 domain
B protein was added to the duplex DNA solutions, and fluorescence
lifetime data were taken at 0, 1, 10, and 20 eq of added protein.
Fluorescence Lifetime Data Analysis--
The fluorescein
lifetime data were analyzed by using standard least-squares methods.
The decays were assumed to conform to Equation 2 (33), in which
D(t) is the "true" decay function, R(t) is the instrument response function,
determined experimentally to have a full width at half-maximum of 270 ps by scattering ps laser pulses at the MCP-PMT, and C and
to are constants.
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(Eq. 2)
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The CR(t + to) term
provides a correction for laser scatter, which was very difficult to
eliminate completely, but always accounted for less than 1% of the
detected photons. This correction assumes that the contribution of the
scatter to the decay curve is identical to the instrument response
function, that is, that all of the scatter arrives at the detector at
t = 0. The constant to serves
simply to set the zero of time. Both single and double exponential fits
were performed (see below). For a single exponential fit, A
exp(
t/
), the total number of fitted
parameters would be four: A, amplitude of decay;
,
lifetime; C, amplitude of scattered light; and
to, zero time. The least-squares algorithm
employed was a modified version of the Levenberg-Marquardt routine
provided by Numerical Recipes (34).
The fluorescence lifetimes obtained from the fitting of the decay
curves were used to calculate the FRET efficiencies and distances. The
efficiency of energy transfer between donor and acceptor can be
calculated by using Equation 3, where E is the efficiency of
the energy transfer,
DA is the fluorescence lifetime of
the donor in the presence of acceptor, and
D is the
fluorescence lifetime of the donor in the absence of acceptor (16).
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(Eq. 3)
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The distance between donor and acceptor, R, can be
calculated from the FRET efficiency, E, according to
Equation 4, where Ro is a characteristic
distance related to the properties of the donor and acceptor at which
50% of the energy is transferred (16).
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(Eq. 4)
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An Ro value of 45 Å determined for
fluorescein and rhodamine (20) was used to calculate R.
Modeling the HMG1 Domain B Complex with Cisplatin-modified
DNA--
Fig. 2 displays the geometric model used to evaluate a bend
angle for the HMG1 domain B complex with cisplatin-modified DNA. This
model is based on one (20, 22) where the 20-bp oligonucleotide is
represented as a cylinder with the donor and acceptor dyes extending
from the ends. Vectors describing the positions of the dyes are
determined as the cylinder bends and used to calculate the distance
between the dyes. The hinge point of the bend is chosen as the midpoint
of the central axis of the cylinder and is designated as the origin in
(x, y, z) space.
The molecular modeling program QUANTA (Molecular Simulations, Inc.) was
used to determine the initial positions of the donor and acceptor dyes.
The first aspect examined in the model was the distance of the dyes
relative to the center of the cylinder. Models of the two fluorescent
dyes with their six-carbon linker chains were constructed and minimized
by using the CHARMM routine contained in QUANTA. The measured distance
from the center of the fluorophores to the end of the linker chain was
~1.45 nm for both dyes. If the radius of the DNA is assumed to be 1.0 nm, then the perpendicular distance of the dyes from the center of the cylinder will be 2.45 nm. The dyes may bend in toward or away from the
center of the cylinder, as shown in Fig.
2. By keeping the 1.0-nm cylinder radius
constant and allowing the 1.45-nm dye vector to bend, one can determine
the components of the 1.45-nm vector that are perpendicular and
parallel to the central cylinder axis.

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Fig. 2.
Geometric model used to calculate bend angle
( ) for fluorescence lifetime data. The
cylinder represents the 20-bp oligonucleotide. The dyes, labeled
Rh and Fl for rhodamine and fluorescein,
respectively, are allowed to bend toward and away from the normal to
the cylinder axis at some angle, . The model requires the
determination of both the parallel ( ) and perpendicular ( )
components of the dye vectors. The definition of the (x,
y, z) axes used in the model is shown here.
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The second crucial aspect of this model is the position of the
fluorescent dyes relative to the bend in the DNA. In order to determine
these positions, a B-form DNA model of the 20-bp oligonucleotide was
constructed in QUANTA with a rhodamine dye docked onto the 5' end of
the top strand and a fluorescein dye docked onto the 5' end of the
bottom strand. A cisplatin fragment analog,
cis-{Pt(NH3)-(NH2(CH2)nCH3)}2+,
was docked to the N-7 positions of the guanines in the top strand to
mark the location of the 1,2-intrastrand d(GpG) cross-link and the
corresponding DNA bend. Fig. 3 shows this
model, which displays the positions of the fluorescent dyes relative to
the cisplatin directed bend when projected down the helix axis. This view represents a slice of the cylinder in an (x,
z) plane with the hinge point, (0, 0), lying in the center
of the helix. The cisplatin adduct is defined to lie on the
x axis. Measuring the angle between the vectors describing
the position of the dyes and the x axis allows the
components of these vectors along the x and z
axes to be determined. In this case, the angle between the rhodamine
vector and the x axis is 70°, and the angle between the
rhodamine and fluorescein vectors is 200°. The latter value is in
relatively good agreement with the value of 227° used by others
(20).

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Fig. 3.
A view down the helix axis of a 20-bp B-form
DNA helix. Rhodamine is attached to the 5'-end of the top strand,
and fluorescein is attached to the 5'-end of the bottom strand. A
cisplatin analog is docked onto the guanines of the top strand to
indicate the position of the DNA bend. The angles observed between the
dyes and the x axis in this figure are used in the geometric
model to help determine the vectors describing the positions of the
dyes.
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In order to determine the vectors describing the positions of the dyes,
both aspects of the model described above were combined. First, the
perpendicular components of the 1.45-nm dye vectors were determined as
they bend in toward and away from the cylinder. The magnitude of these
components was used along with the angles determined in Fig. 3 to
calculate the components of the dye vectors along the x and
z axes. The component of the vector along the y
axis is the sum of the parallel component of the 1.45-nm dye vector
combined with the length of half the cylinder. In this case, since the
hinge point is located in the center of the helix, its distance to the
top of the cylinder is 3.4 nm (0.34 nm/bp for 10 bp). Once the vectors
were determined for a series of rhodamine and fluorescein angles, the
distance between the two dyes was calculated.
Only certain rhodamine and fluorescein angle pairs will yield the
6.4-nm distance (see "Results and Discussion") determined for the
unplatinated, linear duplex in these FRET experiments. Pairs of
fluorescein and rhodamine angles compatible with the 6.4-nm distance
are revealed by the plot shown in Fig. 4.
Although all of these allowed angle pairs yield a 6.4-nm distance for
the unbent cylinder, some of the positions are preferred by the dyes. Fluorescence anisotropy experiments have indicated that fluorescein tends to have a low anisotropy value, indicative of high rotational freedom, whereas rhodamine tends to have a high anisotropy value, suggestive of an interaction between its dimethylamino groups and the
phosphate backbone of the DNA (17, 20, 35). Thus, those allowed angle
pairs where rhodamine has a negative angle and is bent toward the DNA
may be preferred over those where the dye has a positive angle and is
bent away from the DNA. It is important to keep in mind that these
allowed angles are only average positional preferences. The dyes are
assumed to rotate freely in the FRET equations used above to calculate
distances. In the bend angle calculations described below, the
preferred allowed angle pairs were examined together with positions
that may be less favored.

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Fig. 4.
A plot of the allowed fluorescein and
rhodamine angle pairs for the geometric model using the B-form DNA
parameters and the 6.4-nm distance measured for the unmodified FRET
probe. There are many different allowed angle pairs. Those with
rhodamine having a negative angle and fluorescein having a positive
angle are preferred based on anisotropy studies and are denoted by the
shaded area. These allowed angles are
representative of those average positions in space where a 6.4-nm
distance will be found.
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The bending of the cylinders was performed in (x,
y) space as depicted in Fig. 2 for the allowed rhodamine and
fluorescein angle pairs calculated above. As the cylinder bends, the
position of the rhodamine dye, and hence the vector which describes its position, was kept constant to simplify the model. The bottom half of
the cylinder and the fluorescein dye moves in (x,
y) space. By allowing the cylinder to bend only in an
(x, y) plane, the position of the fluorescein dye
relative to the z axis remains constant. The x
and y components of the fluorescein vector change as the
cylinder bends (Fig. 5). New fluorescein
vectors for each bend angle were calculated from the altered
x and y components. These new vectors were used
with the rhodamine vector to determine the distance between the dyes.
The bend angle that predicts the measured 5.5-nm distance (see below)
between the dyes was thus estimated for the HMG1 domain B protein-DNA
complex.

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Fig. 5.
A schematic drawing of how the fluorescein
dye vector is determined as the cylinder bends. Since the cylinder
bends in (x, y) space, the component of the
fluorescein vector along the z axis will not change with
. The components of the vector along the x and
y axes change as follows: (x'2 + y'2) cos((90 + ) ) (for x
axis) and (x'2 + y'2)
sin((90 + ) ) (for y axis). The angle, , is
equal to the tan 1 (x'/y').
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The above model does not take into account DNA unwinding, which might
accompany platinum or protein binding. In order to do so, the initial
positions of the dyes described in Fig. 3 need to be altered. The
average helical twists found in the x-ray crystal structure and NMR
solution structures for a 1,2-intrastrand d(GpG) cisplatin adduct were
used to alter the initial positions of the dyes from Fig. 3 (6, 7, 36).
The 6.6-nm distance measured for the platinated duplex without protein
was used to calculate the allowed fluorophore angle positions. Those
angle pairs that have the dyes separated by 6.6 nm when the DNA bends
by the angles observed in the x-ray and NMR studies were used in the
geometric model. With these new initial parameters, the distances
between the dyes as the cylinder bends were calculated as described above.
Stopped-flow Kinetic Studies with HMG1 Domain B--
These
experiments were performed with a Hi-Tech SF-61 DX2 double mixing
stopped-flow apparatus at 4 °C. HMG1 domain B was combined with 25 nM unmodified or cisplatin-modified duplex probes labeled
with both fluorescein and rhodamine in 10 mM HEPES, pH 7, 200 mM NaCl. Pseudo-first order kinetic conditions were
maintained by using at least a 5-fold excess of protein in all
experiments. The excitation wavelength was 480 nm. A GG495 glass
cut-off filter and a Wratten gel filter no. 58 (Eastman Kodak) were
placed over the exit to the photomultiplier tube. This filter pair
served to block rhodamine fluorescence and transmit the FRET-induced fluorescein fluorescence change.
Concentration-dependent studies were performed with the
cisplatin-modified probe and HMG1 domain B to determine the rate
constants for the binding and dissociation of this protein-DNA complex. In these experiments, the protein concentration was varied from 0.125 to 1 µM. Multiple shots were taken at each protein
concentration. The observed rate constants for the protein
concentrations were determined by fitting individual traces with the
KinetAssyst software package (Hi-Tech) and averaging the results. The
observed rate constant, kobs, was plotted
against the total protein concentration, [Pt].
The resulting linear plot is described by Equation 5, where kon is the second-order rate constant for
protein binding and koff is the first-order rate
constant for the dissociation of the protein-DNA complex.
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(Eq. 5)
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In these experiments, the least-squares fit of each point was
weighted by the number of shots averaged to determine the
kobs value. The rate constants, determined in
Equation 5, were used to calculate a dissociation constant,
Kd, for the protein-DNA complex according to
Equation 6.
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(Eq. 6)
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RESULTS AND DISCUSSION |
Determination of Kd from Steady-state Fluorescence
Titration Experiments--
Fig. 6 shows
emission spectra from three different fluorescence titration
experiments in which the fluorescein dye was excited at 480 nm and
emission spectra were recorded from 490 to 610 nm. The fluorescein
emission maximum occurs at 520 nm, and the rhodamine emission maximum
at 580 nm. Panel A of Fig. 6 presents the results of titrating Pt/Rh/Fl-ds with increasing amounts of HMG1 domain B. The
inset enlarges the spectral region from 565 to 600 nm. Panel B displays the unplatinated Rh/Fl-ds sample
with increasing amounts of HMG1 domain B, and panel
C shows the platinated Pt/Rh/Fl-ds probe titrated with
distilled, deionized water instead of protein. The results clearly
demonstrate that significant spectral changes occur only when the
platinated probe is titrated with protein.

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Fig. 6.
Emission spectra from fluorescence titration
experiments. A, Pt/Rh/Fl-ds with increasing HMG1 domain
B; B, Rh/Fl-ds with increasing HMG1 domain B; C,
Pt/Rh/Fl-ds titrated with distilled, deionized water. The
inset to A shows an enlargement of the spectral
region from 565 to 600 nm to highlight the increase in rhodamine
fluorescence.
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From the decrease in the fluorescence intensity at 520 nm for the
fluorescein donor and the increase in the fluorescence emission of the
rhodamine acceptor dye at 580 nm with increasing protein concentration
(inset), it is clear that FRET occurs. We ascribe this
result to protein-induced bending of the platinated DNA. The spectral
observed changes were used to determine the dissociation constant of
this protein-DNA complex. The change in fluorescence intensity at 520 nm (
F) is directly related to the concentration of the
protein-DNA complex, assumed to have a 1:1 stoichiometry. Plots of
F versus [Pt] (Fig.
7) were fit by least squares to Equation 1, affording an average dissociation constant of 60 ± 30 nM.

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Fig. 7.
Determination of Kd
from titration data. The data shown are for a single titration
experiment fit to Equation 1. Several experiments were carried out, and
the results averaged to determine the final dissociation constant
value.
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Recently, a series of cisplatin-modified 15-bp oligonucleotides were
reported to have a range of dissociation constants for the HMG1 domain
B complex of 50-1300 nM (37), as determined by gel
mobility shift assays. The value of 60 ± 30 nM
determined here falls within this range, but is ~8 times smaller than
the one previously determined for the identical 20-bp oligonucleotide by gel mobility shift assay (13). This discrepancy is ascribed to
differences in the experimental conditions, the present experiments being performed at a different temperature and with different buffer
conditions than in the gel mobility shift assay.
Duplex End-to-end Distance from Fluorescein Fluorescence Lifetime
Measurements--
Fluorescence emission decay curves were obtained and
the data were analyzed as described above. An example of a fitted
fluorescence emission decay curve is presented in the upper
panel of Fig. 8. The
bottom panel of this figure depicts the weighted
residuals of the fit, (Iobs
Icalc)/
i, where
i is the standard deviation associated with
Iobs,
i = (Iobs)0.5. Most of the weighted
residuals lie within two standard deviations. The fluorescence lifetime
values obtained from fitting the decay curves are presented in Table
I.

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Fig. 8.
Example of a fitted fluorescence emission
decay curve. Data were fitted as described under "Experimental
Procedures." The upper panel shows the fitted
decay curve, and the lower panel depicts the
weighted residuals of the fit.
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The data in Table I show that, for the fluorescein-labeled (Fl-ds),
platinated fluorescein-labeled (Pt/Fl-ds), and rhodamine- and
fluorescein-labeled (Rh/Fl-ds) duplex oligonucleotides, the fluorescein
fluorescence lifetime either remains the same or increases slightly
with increasing concentrations of HMG1 domain B. The data for the
platinated oligonucleotide with rhodamine and fluorescein (Pt/Rh/Fl-ds)
clearly display a different trend. The lifetime of fluorescein in this
probe in the absence of added protein is slightly increased from that
of the Rh/Fl-ds duplex, presumably due to DNA unwinding and bending
upon platination. When increasing concentrations of HMG1 domain B were
added, there was a significant decrease in fluorescence lifetime of the
fluorescein label. This difference in lifetimes is apparent in the
decay curves plotted in Fig. 9, the log
format of which emphasizes the effect. In this figure, the Pt/Fl-ds and
Pt/Rh/Fl-ds probes with no protein are compared with the Pt/Rh/Fl-ds
probe with 20 eq of added HMG1 domain B. The longest fluorescence
lifetime is for fluorescein in the Pt/Fl-ds sample. The lifetime
shortens when the acceptor dye is present, and with 20 eq of HMG1
domain B the Pt/Rh/Fl-ds probe exhibits the shortest fluorescence
lifetime. The changes are consistent with FRET induced by protein
binding and bending the platinated DNA.

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Fig. 9.
Normalized fluorescence lifetime decay curves
for some selected samples. Curves for Pt/Fl-ds, Pt/Rh/Fl-ds, and
Pt/Rh/Fl-ds with 20 eq HMG1 domain B are depicted. Plots are shown in
log scale for clarity.
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The fluorescence lifetime values were used to calculate FRET efficiency
(E) and the distance (R) between the dyes in
these different samples. Table II shows
the results computed by Equations 3 and 4 for the Rh/Fl-ds and
Pt/Rh/Fl-ds probes. As expected from the lifetime studies, the Rh/Fl-ds
samples exhibit only a small increase in FRET efficiency with
increasing protein concentrations, corresponding to a decrease in
distance of only ~0.1 nm, from 6.4 to 6.3 nm. The Pt/Rh/Fl-ds
samples, however, show a dramatic increase in FRET efficiency, from
0.09 to 0.23 with increasing protein concentrations. This result
reflects a change in distance between donor and acceptor of
1.1 nm,
from 6.6 to 5.5 nm. The 17% decrease may be attributed to protein
binding and concomitant DNA bending.
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Table II
FRET efficiency and distance data
Numbers in parentheses are standard deviations in the last significant
digit.
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Most of the fluorescence decay curves were adequately fit to a
single-exponential function. The Pt/Rh/Fl-ds samples containing excess
HMG1 domain B deviated the most from single exponential behavior.
Double-exponential fits were therefore investigated, the results of
which are reported in Table III. The
double-exponential fits at the higher concentrations of protein afford
fluorescence lifetimes of approximately 5.0 and 2.4 ns. One possible
explanation for this behavior is that the lifetimes reflect
contributions from both bound and free DNA (26). In this
interpretation, the shorter lifetime value would represent the protein
bound DNA, and the ~5.0-ns lifetime, the free DNA. Using this value
for the bound DNA, the FRET efficiency increases from 0.09 to ~0.4,
corresponding to a distance change of 1.9 nm and indicating greater DNA
bending (~90-120°). The ~5.0-ns lifetime value for the free DNA,
however, is much longer than any observed for fluorescein in the single exponential fits of the data, making this explanation unsatisfactory. In addition, even in the most extreme cases, the discrepancy between the observed data and the single-exponential fits was fairly small, and
the double-exponential fits improved chi-squared by at most ~40%
over the single exponential fits. Therefore, in the absence of an
adequate explanation for the weakly multiexponential behavior of some
of the decays, we use the single-exponential decay values to calculate
FRET efficiency in all cases.
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Table III
Double exponential fits to data for Pt/Rh/Fl-ds samples with excess
HMG1 domain B
Numbers in parentheses are standard deviations in the last significant
digit.
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Modeling the Bending of the HMG1 Domain B-Cisplatin-modified DNA
Complex--
The distances determined from the FRET lifetime
experiments were used to estimate a bend angle for the protein-DNA
complex. Figs. 2-5 describe the geometric model used along with the
distances determined from the single-exponential fits to compute a
bend angle for the HMG1 domain B complex with cisplatin-modified DNA. In the first case examined, geometric parameters for normal B-form DNA
were used along with the distance determined for the unmodified FRET
probe. The bending of the cylinder, which created a 5.5-nm separation
between the fluorophores, occurred in the range of 90-100°. The
geometric model was then altered to examine the effects of DNA
unwinding on the estimated bend angle. A smaller bend of ~70-85°
obtained when the helical twist and bend angle range from the x-ray
crystal structure were employed is judged to be less reliable because
of duplex-duplex packing interactions in the solid state that render
these values less relevant to the solution structure (6). Using the
average helical twist and bend angle range determined for the NMR
solution structures of duplex DNA containing a 1,2-intrastrand d(GpG)
cisplatin adduct (7, 36), the 5.5-nm distance occurred when the DNA was
bent by ~80-95°. The ~60-80° bend and underwinding of the
platinated DNA duplex push the limits of the geometric model used to
estimate a bend angle from the FRET data. In particular, when compared
with models where the helical twist and bend angles from B-form DNA
were used, the number of allowed fluorescein and rhodamine angles were
significantly fewer. Moreover, none of the angle pairs, which yielded a
6.6-nm distance, was in the preferred position where rhodamine bends toward the DNA. Hence, it is important to examine these estimated bend
angles and see how they compare with ones determined by other techniques.
The ~80-95° range estimated using helical twists and bend angles
from the NMR studies of the 1,2-intrastrand d(GpG) cisplatin adduct
seems reasonable, based on the results of NMR solution studies of DNA
complexes with other HMG- domain proteins (38, 39). In the SRY NMR
structure, the DNA is bent by ~70-80° (39), and for LEF-1, the DNA
is bent by ~117° (38). The bend angle range estimated with our
geometric model falls within the range determined in these NMR studies.
The DNA bending caused by the formation of complexes with HMG-domain
proteins has also been investigated by gel mobility shift assays (12)
and x-ray crystallography.3
The bend angle determined in gel shift studies for the HMG1 domain B
complex with a cisplatin-modified DNA probe was ~65-74° (12). X-ray crystallography shows the bend angle for a cisplatin-modified DNA
probe bound to HMG1 domain A to be ~61°.3 Both of these
values are smaller than what was estimated from our geometric models.
The discrepancy between the present bend angle value and that for other
complexes of HMG-domain proteins with platinated DNA is ascribed in
part to differences in experimental methods. For example, crystal
packing forces can affect the bend angle in an x-ray crystal structure
compared with the FRET studies, where the complex is free in solution.
Another possible contribution to the larger bend angle, estimated here,
could be the different DNA sequence employed. Recent work indicates
that the sequence context surrounding the 1,2-intrastrand d(GpG)
cisplatin adduct modulates the binding affinity of HMG-domain proteins
(37) and may influence the ability of the DNA to bend when an
HMG-domain protein binds (6, 37). In the FRET study, the platinated sequence was TG*G*T, whereas the sequence in
the gel shift assay study was AG*G*C and in the
x-ray study, TG*G*A, where G*
denotes the modified nucleotide. The presence of an additional surrounding A/T base pair may increase the bendability of the DNA used
in the FRET experiment compared with the gel shift study owing to the
fewer number of hydrogen bonds (37). From these considerations, we
conclude that the 80-95°-bend angle range estimated from the
geometric model for these FRET studies is reasonable.
Rate of HMG1 Domain B Binding to Cisplatin-modified DNA:
Stopped-flow Kinetic Experiments--
Stopped-flow kinetic experiments
were performed with both the unmodified and cisplatin-modified DNA
probes containing fluorescent labels. Fig.
10 portrays results from some of these
experiments. In panel A are presented data from
some of the individual shots. Curve 1 shows the
unmodified probe with 500 nM HMG1 domain B. No fluorescence
change is observed for this sample. Curves 2 and 3 exhibit the cisplatin-modified probe in the presence of
188 and 375 nM HMG1 domain B, respectively. Here, a
decrease in the fluorescein fluorescence is observed over time that is
ascribed to protein binding and concomitant bending of the DNA.
Although the protein concentration is always in excess, in order to
maintain pseudo-first order conditions, the two different
concentrations of protein shown in panel A
clearly demonstrate that the rate decrease depends on protein
concentration.

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Fig. 10.
Stopped-flow kinetic experiments with HMG1
domain B. Panel A displays individual shots.
Curve 1 is the unmodified duplex probe with 500 nM HMG1 domain B. Curves 2 and
3 show the cisplatin-modified duplex probe with 188 and 375 nM HMG1 domain B, respectively. Panel
B is a plot of the concentration dependent data fitted to
Equation 6, from which were determined kon and
koff values of 1.1 ± 0.1 × 109 M 1 s 1 and
30 ± 4 s 1, respectively.
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A concentration-dependent study of the observed rate
constants was carried out for the cisplatin-modified probe binding to HMG1 domain B in order to determine the rate constants for binding and
the dissociation of the complex. The results from these experiments are
illustrated in panel B of Fig. 10. A fit of the
data to Equation 5 gives a second-order rate constant for binding,
kon, of 1.1 ± 0.1 × 109
M
1 s
1 and a first-order rate
constant for the dissociation of the protein-DNA complex,
koff, 30 ± 4 s
1
(t1/2 = 0.023 s). The dissociation constant,
Kd, determined from these rate constants using
Equation 6 is 27 ± 4 nM, which is in agreement with
the value determined in the fluorescence titration experiments.
The results of these experiments provide the first information about
the kinetics of HMG-domain proteins binding to cisplatin-modified DNA.
Very little kinetic information is available for HMG-domain protein
binding to DNA in general. Data that are available were obtained
through competition kinetic gel shift experiments, and not by direct
measurement of the binding event, as accomplished in the present FRET
study (40). The kinetics of binding of an HMG-domain protein to
cisplatin-modified DNA is potentially important for understanding its
possible role in sensitizing cells to the drug. Recent experiments
examined the interactions of RPA, a protein involved in DNA damage
recognition in the nucleotide excision repair pathway, and HMG1 to
cisplatin-modified DNA (41). The results of these experiments revealed
that, when both proteins were present, HMG1 selectively bound the
platinated DNA (41). One possible explanation for this result is that
HMG1 binding to cisplatin-modified DNA occurs at a rate faster than
that of RPA binding and that kinetics controls the competition (41). The kon value for HMG1 determined here, which is
near the diffusion limit, is consistent with such an explanation.
In conclusion, the present study illustrates the utility of the FRET
technique for investigating the structure and dynamics of proteins
binding to platinated DNA. The method offers the potential for
measuring distances and binding rates in solution and has an advantage
over x-ray crystallographic and gel mobility shift approaches, which do
not permit solution studies to be carried out over a wide range of
conditions, such as ionic strength or pH. FRET experiments can also be
performed using low concentrations of material, an advantage over NMR
spectroscopy. Finally, it is relatively easy to alter the nature of the
fluorescent label and to examine FRET distance changes with different
oligonucleotide sequences. By varying the length and studying a series
of oligonucleotide probes, more elaborate models may be constructed to
examine changes in the helical parameters of the DNA (20, 42).