Stopped-flow Kinetic Analysis of Replication Protein A-binding
DNA
DAMAGE RECOGNITION AND AFFINITY FOR SINGLE-STRANDED DNA REVEAL
DIFFERENTIAL CONTRIBUTIONS OF kon AND
koff RATE CONSTANTS*
Steve M.
Patrick
and
John J.
Turchi§
From the Department of Biochemistry and Molecular Biology, Wright
State University School of Medicine, Dayton, Ohio 45435
Received for publication, November 13, 2000, and in revised form, March 5, 2001
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ABSTRACT |
Replication protein A (RPA) is a heterotrimeric
protein required for many DNA metabolic functions, including
replication, recombination, and nucleotide excision repair (NER). We
report the pre-steady-state kinetic analysis of RPA-binding DNA
substrates using a stopped-flow assay to elucidate the kinetics of DNA
damage recognition. The bimolecular association rate,
kon, for RPA binding to duplex DNA substrates
is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA
adduct, and least for an undamaged duplex DNA substrate. RPA displays a
decreased kon and an increased koff for a single-stranded DNA substrate
containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an
undamaged DNA substrate. The kon for
RPA-binding single-stranded polypyrimidine sequences appears to be
diffusion-limited. There is minimal difference in kon for varying length DNA substrates;
therefore, the difference in equilibrium binding affinity is mainly
attributed to the koff. The
kon for a purine-rich 30-base DNA is reduced by
a factor of 10 compared with a pyrimidine-rich DNA of identical length.
These results provide insight into the mechanism of RPA-DNA binding and
are consistent with RPA recognition of DNA-damage playing a critical
role in NER.
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INTRODUCTION |
The heterotrimeric
RPA1 protein consists of 70-, 34-, and 14-kDa subunits and is involved in many DNA metabolic
functions. These functions involve both the interaction of RPA with
other proteins and binding to DNA. The equilibrium DNA binding affinity of RPA has been well characterized for single-stranded DNA substrates and reveals a sequence and length dependence. RPA displays a higher affinity for pyrimidine-rich DNA versus purine-rich DNA
sequences, and RPA displays an increased affinity as DNA length
increases (1). RPA duplex DNA binding has been observed but appears to be a result of DNA denaturation and the subsequent binding to the
single-stranded structure of the DNA (2, 3). RPA has also been shown to
preferentially bind to duplex-damaged DNA compared with undamaged DNA
(4, 5). This preferential binding correlated well with the ability of
RPA to denature the duplex-damaged DNA (6). Whether RPA can actually
bind duplex DNA without denaturing the duplex structure remains to be determined.
Nucleotide excision repair (NER) is the major cellular pathway
for removing bulky DNA adduct damage and helps maintain genomic stability. NER involves multiple protein components and is divided into
four distinct steps. These steps include damage recognition, incision,
displacement, and resynthesis and ligation. The rate-limiting step of
NER is the recognition of the damaged DNA (7, 8). This recognition
process is poorly understood in eukaryotes but appears to involve the
RPA, XPA, XPC·hHR23B, and potentially the XPE proteins (7, 9). All
four proteins have been shown to preferentially bind to damaged DNA,
and the RPA·XPA complex has been shown to act synergistically
to bind damaged DNA (4, 5, 10-14). The XPC·hHR23B protein complex
has been suggested to be the "initiator" of global genomic repair
(12), although some controversy still exists as to the exact role of
this protein in NER (15). Slower rates of repair were observed when
XPC·hHR23B was the first protein to bind the damaged DNA while
preincubation of the RPA·XPA complex with damaged DNA resulted in
faster repair, consistent with the RPA·XPA complex initiating repair
(15). Our data extend this model and suggest that, once localized
denaturation occurs at the damaged DNA site, RPA binds to the undamaged
DNA strand to protect this strand from incision and directs the XPG and
XPF·ERCC1 proteins to cleave the damaged DNA strand 3' and 5',
respectively (6). This model is also supported by RPA binding polarity
and the positions of the known protein·protein interactions (14,
16-21).
The biochemical process of distinguishing damaged DNA from the vast
background of undamaged DNA still remains to be elucidated, because the
affinities of the individual proteins for damaged DNA cannot account
for the preference required. It appears that DNA adducts that distort
the duplex DNA structure to the greatest degree are repaired more
efficiently than those adducts that cause the least distortion (7, 22).
The protein components of NER may recognize the thermal instability of
the duplex DNA structure created by the DNA adduct and initiate repair
(23). DNA adducts that cause the greatest distortion may only require
the RPA·XPA complex, whereas non-distorting lesions may require the
XPC·hHR23B protein followed by the recruitment of the RPA·XPA
complex. Transcription coupled repair may also play a role in the
repair of non-distorting adducts, which may help the coordinated action
of the XPC·hHR23B and RPA·XPA proteins to account for the
discrimination required for the DNA repair process. Understanding the
kinetics of the protein factors in damaged DNA binding should provide
insight into the mechanism of DNA damage recognition.
Here, we report the use of a stopped-flow fluorescence assay to measure
the pre-steady-state kinetics of RPA binding to various DNA substrates.
The fluorescence assay relies on the intrinsic fluorescence of RPA and
subsequent "quenching" when RPA binds to a DNA substrate (24, 25).
RPA displays a decreased kon and increased
koff for a duplex substrate containing a
1,2d(GpG) cisplatin-DNA adduct compared with a duplex containing a
1,3d(GXG) cisplatin-DNA adduct. In support of RPA binding to the
undamaged DNA strand during NER, we show that RPA displays a decreased
rate of association and an increased rate of dissociation for a
single-stranded DNA substrate containing a single 1,2d(GpG)
cisplatin-DNA adduct compared with the undamaged DNA. The difference in
affinity for DNA length appears to be manifested mainly from a
difference in the rate of dissociation, koff.
The kon for pyrimidine-rich sequences was
determined to be near diffusion-limited but was reduced by a factor
greater than 10 for a purine-rich DNA sequence of the same length.
These results will be discussed with respect to DNA damage recognition
and nucleotide excision repair.
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EXPERIMENTAL PROCEDURES |
Materials--
DNA oligonucleotides were purchased from
Integrated DNA Technologies, Inc. (Coralville, IA), including the
5'-fluorescein-labeled DNA. All oligonucleotides were purified by 15%
polyacrylamide, 7 M urea preparative sequencing gel
electrophoresis. Cisplatin was purchased from Sigma Chemical Co. (St.
Louis, MO), and mung bean nuclease was purchased from Life
Technologies Inc. The SP5.2 DNA sequence is
5'-GGGGAAGGAAGAGGCCAAGAAGGAGAGGGG-3'.
Protein Purification--
Recombinant RPA was purified as
described using the expression vector kindly provided by Marc Wold with
the following modifications (26). During the RPA purification, protein
was monitored via absorbance at 280 nm, and RPA DNA binding activity
was assessed in EMSAs using labeled dT30 ssDNA. The supernatant from 2 liters of an
isopropyl-1-thio-
-D-galactopyranoside-induced culture
(15 ml) was applied to a 20-ml Affi-gel blue column equilibrated in HI
buffer (30 mM HEPES, pH 7.8, 0.01% Nonidet P-40, 0.25 mM EDTA, 1 mM dithiothreitol, and 0.25%
inositol) (26) supplemented with 0.1 M KCl. The column was
sequentially washed with HI buffer containing 0.8 M KCl and
0.5 M NaSCN. RPA was eluted with HI buffer containing 1.5 M NaSCN. The peak fractions (30 ml) were pooled based on
the absorbance at 280 nm and applied directly to a 5-ml hydroxylapatite column equilibrated in HI buffer. After washing with HI buffer, RPA was
eluted in HI buffer containing 100 mM potassium
Pi. The DNA binding pool of RPA (15 ml) was loaded
onto a 2-ml Q-Sepharose column equilibrated in HI buffer and washed
with HI buffer containing 0.1 M KCl. RPA was eluted with HI
buffer using a linear gradient from 0.1 to 1 M KCl. The
peak fractions of RPA were pooled and dialyzed in HI buffer.
Platination and DNA Purification--
Platination reactions were
performed as previously described (5, 6). Briefly, the SP10.2 DNA
substrate (5'-TTTTTTGGTTTTTTT-3') was either left undamaged or treated
at a 3:1 molar ratio of cisplatin to GG sites in buffer containing 1 mM NaHPO4 (pH 7.5) and 3 mM NaCl at
a maximum concentration of 1 pmol/µl DNA for about 20 h at
37 °C in the dark. The platinated substrate was purified on a 18%
preparative sequencing gel to separate the undamaged DNA from the DNA
reacted with cisplatin and eluted and ethanol-precipitated (27). The
duplex cisplatin-damaged 1,2d(GpG) and 1,3d(GXG) 30-bp DNA substrates
were purified as previously described to ensure 100% platination and
less than 1% single-stranded DNA contamination (6).
Stopped-flow Kinetic Experiments and Data
Analysis--
Stopped-flow kinetic traces were obtained using a
SX.18MV stopped-flow reaction analyzer (Applied Photophysics). Equal
volumes of RPA and DNA in buffer containing 20 mM HEPES (pH
7.8), 2 mM dithiothreitol, 0.001% Nonidet P-40, 50 mM NaCl, and 2 mM MgCl2 from
separate syringes were rapidly mixed at 24 °C. Fluorescence was
measured following excitation at 290 nm (0.5 mM slit width) using a 350-nm long-pass cut-on filter (LG-350 from Corion, Franklin, MA). Constant RPA (6.25 nM final concentration) and varying
DNA concentrations (31.25-200 nM) starting at a 5-fold
excess of DNA were used to achieve pseudo-first order kinetics (28).
The traces depicted for each concentration of DNA represents the
average of 8-12 individual shots. The kinetic data were fit using
Pro-K software (Applied Photophysics) to calculate the observed rate, kobs. The single-stranded DNA data fit to a
single-exponential decay. Assuming a simple mechanism of
single-stranded DNA binding, RPA + DNA
RPA·DNA then,
kobs = kon [DNA] + koff, where the slope is the bimolecular
association rate, kon, and the
y-intercept is the rate of dissociation,
koff (28). The individual points on each graph
represent the average and standard deviation of a minimum of three
independent experiments. Rate constants were determined by linear
regression analysis using SigmaPlot (Jandel). The data are presented in
Table I as the average and standard error of the fit of each
line. Nearly identical values were obtained when each experiment was
graphed independently, and rate constants were calculated and then the
average and standard deviation calculated (analysis not shown). Results
obtained for RPA-binding duplex DNA fit to a double-exponential decay.
Both the fast and slow phase of RPA binding to the duplex DNA substrate
were plotted versus DNA concentration to obtain rate
constants. To confirm the calculated koff
values, RPA was prebound to a 5'-fluorescein-labeled DNA at room
temperature. The complex was then mixed with an excess of
single-stranded unlabeled competitor DNA, typically 100-500 nM dT30. The reactions were excited at 495 nm,
and emission was monitored using a 530-nm long-pass cut-on filter (OG
530 from Edmund Industrial Optics, Barrington, NJ). RPA binding to the fluorescein-labeled DNA resulted in either an increase or decrease in
fluorescence dependent on DNA length and sequence. The change in
fluorescence as RPA dissociated from the fluorescein-labeled DNA was
monitored over time and was used to calculate the
koff. The experimentally determined
koff values were consistent with the positive
y-intercept values obtained using shorter DNA substrates. For the single-stranded platinated SP10.2 DNA and the duplex
substrates, the y-intercept value determined from the
kon experiments was used as the
koff value. For Table I, the
KD values were calculated using the
kon values and either the
koff values determined experimentally or from
the y-intercept values using the following equation:
KD = koff/kon (28-30).
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RESULTS |
Kinetics of RPA-binding Duplex-undamaged and
Cisplatin-damaged DNA--
Intrinsic fluorescence has been monitored
for a variety of proteins via tryptophan or aromatic amino acids within
active sites or DNA binding sites (24, 29, 31). Here, we have employed the quenching of RPAs intrinsic tryptophan fluorescence upon binding DNA to analyze the kinetics of the interaction with DNA. We have previously shown that RPA preferentially binds to duplex DNA containing cisplatin intra-strand DNA adducts over undamaged DNA (5, 6). We also
showed that RPA displayed better binding to DNA containing the
1,3d(GXG) cisplatin-DNA adduct than DNA with the 1,2d(GpG) adduct (6).
This binding is consistent with the distortion in the duplex structure
generated by the two different types of cisplatin-DNA adducts (32, 33).
To assess the kinetics of RPA binding to duplex DNA containing both
types of cisplatin adducts, stopped-flow analysis was performed using
duplex DNA substrates containing either a single 1,2d(GpG) or 1,3d(GXG)
adduct. The quenching of the intrinsic tryptophan fluorescence of
constant amounts of RPA was monitored over time at a variety of DNA
concentrations. The excitation wavelength was set at 290 nm, and the
emission was monitored via a 350-nm cut-on filter. All reactions were
performed in buffer containing 50 mM NaCl and 2 mM MgCl2 as described under "Experimental
Procedures." The results presented in Fig.
1A reveal a
DNA-dependent fluorescence quenching effect with the duplex DNA substrate containing a 1,2d(GpG) cisplatin-DNA adduct. Control traces with buffer or DNA alone were performed and were identical (data
not shown). The traces presented in each figure represent the
fluorescence above this baseline value. The trace obtained with RPA
alone (top trace) provides the initial fluorescence from which quenching was monitored. Each trace was fit to a
double-exponential decay (thick line), and the residual
values were presented in the panel beneath each trace. The
data were not consistent with a single-exponential decay (not shown).
Fig. 1B represents the trace of RPA with duplex DNA
containing a 1,3d(GXG) cisplatin-DNA adduct. Again, the top
trace was obtained with RPA alone, and the bottom trace
was obtained by mixing RPA with DNA.

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Fig. 1.
Kinetics of RPA-binding duplex
cisplatin-damaged DNA substrates. Kinetic traces were measured at
an RPA concentration of 6.25 nM and a DNA concentration of
50 nM in buffer containing 50 mM NaCl and 2 mM MgCl2. The reactions were excited at 290 nm,
and fluorescence was monitored via emission at 350 nm. The control
trace is the RPA and buffer mixed (top trace). The buffer
alone and DNA/buffer mix were used as blank controls (not shown). There
is a time- and DNA-dependent decrease in RPA intrinsic
fluorescence (bottom trace). Each trace is an average of
8-12 measurements at each DNA concentration, and the kinetic traces
were fit to a double-exponential decay. A, analysis of RPA
binding to DNA containing a 1,2d(GpG) cisplatin adduct resulted in
kobs values of 4.4 s 1 and 0.14 s 1 for the fast and slow rates, respectively.
B, analysis of RPA binding to DNA containing a 1,3d(GXG)
cisplatin-adduct was performed under identical conditions. The results
revealed kobs values of 4.1 s 1 and
0.14 s 1 for the fast and slow phase of the reaction,
respectively. The residual values obtained for each fit of the data are
presented in the panel below the kinetic traces.
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The observed rate of quenching, kobs, for the
fast phase of quenching was plotted versus the DNA
concentration for both the 1,2d(GpG) (open circles) and the
1,3d(GXG) (filled circles) DNA adducts. The results revealed
a linear relationship with the slope being equal to the
kon and the y-intercept equal to the
koff (Fig. 2A) (28, 29). The
kon for the duplex 1,2d(GpG) DNA substrate was
0.022 ± 0.002 nM
1 s
1, and
the koff was 3.39 ± 0.55 s
1.
The kon for the duplex 1,3d(GXG) DNA substrate
was 0.062 ± 0.004 nM
1 s
1
with a koff of 2.21 ± 0.26 s
1. The calculated KD values (Table
I) for the two substrates reveal more
than a 4-fold difference in affinity. The kon
for the duplex undamaged DNA substrate was 0.013 ± 0.002 nM
1 s
1 with a
koff of 9.48 ± 0.41 s
1,
which provides about a 5-fold difference in KD value between the duplex undamaged and 1,2d(GpG) cisplatin-DNA substrates (data not shown). The data suggest a slower association rate and faster
dissociation rate for the duplex undamaged DNA compared with the
cisplatin-damaged DNA substrates, consistent with preferential binding
of RPA to duplex damaged DNA (5, 6). The data also demonstrate a faster
association and slower dissociation of RPA for the duplex 1,3d(GXG)
substrate compared with the duplex 1,2d(GpG) cisplatin-DNA substrate.
This is consistent with the distortion in the duplex generated by the
1,3d(GXG) cisplatin-DNA adduct, which causes about a 2-4 base
localized denaturation, whereas the 1,2d(GpG) cisplatin-DNA adduct
causes no disruption in the hydrogen bonds but decreases the melting
temperature of the duplex by 9 °C (32-34). The single-stranded
structure in the duplex 1,3d(GXG) DNA substrate most likely promotes
the faster association and slower dissociation of RPA compared with the
1,2d(GpG) adduct.

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Fig. 2.
Determination of the kinetic constants for
RPA-binding duplex cisplatin-damaged DNA substrates. A,
the observed rate constants for the fast phase of association were
plotted versus DNA concentration for the 1,2d(GpG)
(open circles) and 1,3d(GXG) (filled circles)
cisplatin-damaged DNA and fit to a straight line. The slope
of the line provides the bimolecular rate constant,
kon, and the y-intercept provides the
rate of dissociation, koff. B, the
kobs values for the slow phase of RPA
association were plotted for both the 1,2d(GpG) (open
circles) and 1,3d(GXG) (filled circles) DNA substrates.
Each point represents the average of three individual experiments, and
the error bars represent the standard deviation.
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The observed rate constants for the slow phase (ks)
of RPA quenching were also plotted versus DNA concentration
(Fig. 2B) and, similar to the fast phase, the rate increased
with DNA concentration. We considered a general two step process where RPA binding to DNA is followed by an isomerization of the complex such
that increased quenching is observed in Model 1 (represented by
Reaction 1) as follows.
This model predicts a hyperbolic curve of
kobs versus DNA concentration and
the data were fit to Eq. 1 (35).
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(Eq. 1)
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Analysis revealed ks values of 2.27 s
1 for DNA containing a 1,3d(GXG) and 0.27 s
1 for DNA containing the 1,2d(GpG) cisplatin adduct.
This demonstrates that the second transformation is faster for the DNA
containing a 1,3 d(GXG) adduct. The k-s values obtained
were near zero, indicating a negligible reverse reaction. To confirm
the model, the individual traces were modeled using ProK software
(Applied Photophysics). The analysis revealed a poor fit of the
individual traces (data not shown). Therefore, we considered a more
complex model that would still be consistent with a double-exponential
fit of the data. Considering the data demonstrating that RPA can
completely denature duplex DNA molecules and the irreversible nature of
the second step, these processes were incorporated in the model.
Model 2 (represented by Reaction 2) involves the irreversible
denaturation of double-stranded DNA to generate an RPA·ssDNA complex
and free single-stranded DNA. The free single-stranded DNA can then
bind a second molecule of RPA. The denaturation step was modeled as a
transparent reaction with no change in fluorescence, and the binding of
the second molecule of RPA to the free single-stranded DNA gives the
same degree of tryptophan quenching. The values of
ks and k
s obtained from
the double-exponential fit of the data are therefore a combination of
k2, k3, and
k
3 from the new model. Fitting the data to
Model 2 revealed that the denaturation step was extremely slow and a
rapid third step, which represents binding of RPA to the
single-stranded DNA. Therefore, the values of ks
represent the rate of denaturation, because this limits the final step
of binding of the second molecule of RPA. This result is
consistent with the decreased thermal stability of the 1,3d(GXG)
compared with the 1,2d(GG) cisplatin adduct, and our previous results
demonstrating RPA binding correlates with the ability to denature
duplex DNAs (6). Analysis of the individual traces using Model 2 yielded excellent fits, which were indistinguishable from fitting the
data to the simple A>B>C double-exponential model that was used to
generate kobs values for each trace. In
addition, plots of the residual values obtained using Model 2 indicated
an excellent overall fit of the data (data not shown).
Kinetics of RPA-binding Cisplatin-damaged Single-stranded
DNA--
Previously, we have shown that RPA binding is inhibited on a
24-mer single-stranded cisplatin-damaged DNA substrate compared with
the undamaged DNA substrate (6). To determine the effect of cisplatin
on the kinetics of RPA binding, stopped-flow kinetic analysis was
performed using a single-stranded 15-base pyrimidine-rich DNA substrate
(SP10.2) in the presence or absence of a 1,2d(GpG) cisplatin-DNA adduct
(Fig. 3A). The kinetic trace
of RPA binding the single-stranded damaged DNA is presented and
illustrates a DNA-dependent fluorescence-quenching effect
(the top trace represents the RPA alone, and the
bottom trace is the RPA mixed with the DNA). Fig.
3B shows the graph of the kobs
plotted versus DNA concentration for the undamaged
(filled circles) and cisplatin-damaged DNA (open circles). The kon for the undamaged DNA was
determined to be 1.71 ± 0.08 nM
1
s
1 with a y-intercept or
koff of 1.57 ± 9.5 s
1. The
cisplatin-damaged DNA resulted in a kon of
0.75 ± 0.04 nM
1 s
1 and a
koff of 23.1 ± 4.2 s
1. The
cisplatin adduct results in a rate of association ~2.5 times slower
and dissociates significantly faster when compared with the undamaged
DNA, resulting in an approximate 20-fold difference in
KD. This data supports our previous model that RPA binds the undamaged DNA strand during NER (6). Considering the large
error in the koff value obtained for the
undamaged pyrimidine-rich single-stranded DNA, the value was determined
using the dissociation kinetics experiments described under
"Experimental Procedures" (data not shown). The value calculated
from these studies revealed a koff value of
2.5 ± 0.1 s
1, consistent with the data
presented above.

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Fig. 3.
Stopped-flow analysis of RPA-binding
undamaged and cisplatin-damaged single-stranded DNA. A,
the kinetic traces for RPA binding to the single-stranded DNA
containing a 1,2d(GpG) cisplatin adduct are shown. The control trace is
represented by the top trace for the RPA/buffer mix. DNA
(31.25 nM) was mixed with the RPA (6.25 nM),
and intrinsic quenching was monitored over time. The traces for
single-stranded DNA fit to a single-exponential decay, and the
kobs value for this particular fit was
calculated to be 46.4 s 1. The residual values for the fit
are provided in the panel below the kinetic traces.
B, the observed rate constants plotted versus DNA
concentration provided a linear fit for both DNA substrates. Each point
represents the average of three individual experiments, and the
error bars represent the standard deviation.
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Kinetics of RPA-binding oligo(dT)30, dT15,
and dT12 DNA--
The characterization of RPA equilibrium
DNA binding has been extensively studied and suggests a clear
difference in affinity between DNA length and sequence (1, 24, 25). It
has previously been shown that RPA displays a decreased affinity for
substrates as the DNA length decreases (1, 24). The difference in
affinity has been suggested to range about two orders of magnitude from a dT10 to a dT50 oligonucleotide (1, 24). To
assess the kinetics of this difference in affinity, RPA stopped-flow
kinetic analysis between varying length pyrimidine DNA was performed. A
DNA concentration-dependent fluorescence-quenching effect
was observed for each DNA substrate (data not shown). Typically, the
percent quenching ranged from 40% to 60% for the single-stranded DNA,
consistent with published data (25). The rate observed at different DNA
concentrations, kobs, was plotted
versus the DNA concentration and yielded a linear graph with
the slope being the kon and the
y-intercept being the koff (Fig.
4) (28, 29). The
kon for the dT30 single-stranded DNA
(filled circles) was determined to be 2.14 ± 0.08 nM
1 s
1, which suggests that the
association is near diffusion limited. The negative value obtained from
the y-intercept suggests a slow dissociation,
koff. The relatively high
kobs values and slow dissociation of the complex
results in large errors associated with the koff determinations. A small change in slope and therefore
kon can result in a dramatic change in
y-intercept. The kon for the
dT15 single-stranded DNA (open circles) was
determined to be 1.81 ± 0.07 nM
1
s
1 with a negative value for the y-intercept.
The difference in the affinity of RPA for a dT30 and
dT15 DNA substrate has been suggested to be greater than
5-fold (1). The difference in the kon for a
single-stranded dT30 and a dT15 substrate is
minimal, suggesting the difference in affinity between the two is
mainly manifested as a difference in the koff.
Decreasing the DNA length from a dT15 to a dT12
also results in about a 5-fold difference in RPA binding affinity (1).
The data for the dT12 single-stranded DNA (open
triangles) resulted in a kon of 1.61 ± 0.09 nM
1 s
1 and a
koff value of 1.46 ± 10 s
1.
These data demonstrate a minimal difference in the rate of association for varying length DNA but support the hypothesis that the rate of
dissociation is the main attribute in the difference in affinity observed with these substrates. However, considering the large errors
associated with these values, it was necessary to design another assay
by which to determine dissociation rate.

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Fig. 4.
Stopped-flow kinetic analysis of RPA-binding
single-stranded dT30, dT15, and
dT12 DNA. Kinetic traces were measured at a constant
RPA concentration of 6.25 nM and varying DNA concentrations
from 31.25 to 200 nM for each substrate. Each trace used
was an average of 8-12 measurements at each DNA concentration, and the
kinetic traces fit to a single-exponential decay. The observed rate
constants (kobs) were then plotted
versus DNA concentration and fit to a straight line. Data
obtained for RPA binding a single-stranded dT30
(filled circles), single-stranded dT15
(open circles), and for single-stranded dT12
(open triangles) DNA substrates are presented. Each point
represents the average of four separate experiments, and the
error bars represent the standard deviation.
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RPA·DNA Dissociation Kinetics--
To measure the rate of RPA
dissociation from DNA substrates that resulted in negative
y-intercept values, a fluorescein based stopped-flow assay
was developed. Fluorescein-labeled DNA was used as the probe with an
excitation wavelength of 495 nm and then emission monitored using a
530-nm cut-on filter. RPA binding to the fluorescein-labeled DNA
results in an increase or decrease in fluorescence that is dependent on
the DNA sequence and length. Fig. 5 shows
the result of koff determination for
single-stranded dT12 DNA. RPA binding to the pyrimidine
dT12 DNA results in a decrease or quenching of the
fluorescence. The two top traces represent the controls with
either buffer alone or fluorescein DNA, which upon giving a
fluorescence signal was reset to the buffer or zero baseline. RPA was
preincubated with the fluorescein-DNA at room temperature, and then the
RPA/fluorescein-DNA was mixed with 100-500 nM unlabeled
competitor dT30 DNA and fluorescence monitored over time
(bottom trace). The fluorescence at time zero is quenched
from the relative fluorescence given by the DNA alone. As RPA
dissociates from the fluorescein-DNA, an increase in fluorescence is
monitored over time. This trace can be fit to a single-exponential rise
(thick line). A plot of the residual values obtained from the fit is presented in the lower panel. The results yielded
a koff of 8.60 ± 0.8 s
1 for
the dT12 DNA substrate and considering the previously
determined kon of 1.61 ± 0.09 nM
1 s
1 (Fig. 4), a
KD value of 5.34 nM was calculated. The experimentally determined koff values and
calculated KD values for the other DNA substrates
are presented in Table I. The results demonstrate that the difference
in RPA equilibrium binding affinity for varying length DNA is
manifested mainly by the koff with minimal
differences in the kon. The duplex
cisplatin-damaged DNA substrates result in a faster
kon and slower koff
compared with the duplex undamaged control DNA, consistent with RPA
being involved in the DNA damage recognition process.

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Fig. 5.
Stopped-flow kinetics measuring the
koff of RPA using fluorescein-labeled
single-stranded dT12 DNA. RPA binds
fluorescein-labeled dT12 DNA and results in a decrease or
quenching of the fluorescence emission (excitation at 495 nm and
fluorescence measured >530 nm). The control traces show buffer alone
(top trace), 1 nM fluorescein-labeled DNA and
buffer after resetting the relative fluorescence to zero (top
trace, overlapping with buffer-alone trace), and 1 nM
fluorescein-labeled DNA/1 nM RPA preincubated and then
mixed with competitor dT30 DNA (100 nM)
(bottom trace). At time zero, the relative fluorescence
signal is quenched, but the addition of single-stranded
dT30 competitor DNA resulted in an increase in the
fluorescence emission over time, indicating the dissociation of RPA
from the fluorescein-labeled DNA. The trace was fit to a
single-exponential fit and gave a koff value of
8.60 s 1. The residual values for the fit of the trace are
presented in the panel below the kinetic traces.
|
|
The single-stranded DNA binding data support the hypothesis that
RPA-stable DNA binding is maintained only on DNA substrates longer than
15 bases. Recently, the 34-kDa subunit of RPA was found to have a DNA
binding domain (36), and the crystal structure of the main DNA binding
domains within the 70-kDa subunit reveals that RPA encompasses 8 bases
of DNA (37). This data suggest that other regions of the 70-kDa
subunit, possibly the C terminus and maybe the 34-kDa subunit need to
contact DNA to form a stable interaction on DNA, because the
length-dependent binding affinity appears to be manifested
by the rate of dissociation of the RPA·DNA complex. Stopped-flow
analysis should provide insight into the mechanism of RPA binding and
provide kinetic data to explain the difference in RPA-binding affinity
for various DNA substrates.
Kinetics of RPA-binding Purine-rich DNA--
RPA has also
been shown to display sequence specific binding with higher affinities
for DNA with pyrimidine sequences (1, 25). The equilibrium DNA binding
reveals about a 50-fold difference in affinity for pyrimidine-rich
versus purine-rich oligonucleotides (38). To assess the
kinetics between pyrimidines and purines, stopped-flow kinetic analysis
was performed using a single-stranded 30-base purine-rich DNA
substrate, SP5.2 (Fig. 6). The sequence of SP5.2 is provided in the "Materials" section under
"Experimental Procedures." The graph of the
kobs values plotted versus DNA
concentration is shown. The kon for RPA binding
SP5.2 was determined to be 0.20 ± 0.01 nM
1 s
1 with a negative
y-intercept value, again indicating a slow dissociation. The
kon for RPA binding the purine-rich SP5.2 DNA is
about 10 times slower when compared with the pyrimidine
dT30 DNA substrates. The koff for
this purine substrate was determined using the fluorescence competition
assay and was determined to be 0.18 ± 0.05 s
1
(Table I). This data demonstrates a 30-fold difference in
KD values between a dT30 DNA substrate
and a 30-base purine-rich DNA substrate. A random DNA sequence with
similar percentages of purines and pyrimidines results in a
kon between the purine-rich and pyrimidine DNA
rates (data not shown). This suggests that the difference in RPA
equilibrium binding observed between purines and pyrimidines is mainly
attributed to the association rate with minimal effect of the rate of
dissociation.

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|
Fig. 6.
Stopped-flow analysis of RPA-binding
single-stranded purine-rich SP5.2 DNA. Kinetic traces were
measured at a constant RPA concentration of 6.25 nM and
increasing DNA concentrations ranging from 31.25 to 200 nM.
Each trace used was an average of 8-12 measurements at each DNA
concentration, and the kinetic traces fit to a single-exponential
decay. Each point represents the average of four individual
experiments, and the error bars represent the standard
deviation.
|
|
 |
DISCUSSION |
RPA is a critical protein component that is required for NER and
DNA replication. In both processes, RPA is involved in the early
enzymatic stages. For DNA replication, RPA stimulates polymerase
/primase priming of the DNA and stabilizes the single-stranded DNA
generated from helicase-catalyzed strand separation (39). In
NER, RPA is involved in the initial stages prior to incision and
possibly plays a key role in the DNA damage recognition step (7, 8,
14). The DNA binding activity of RPA is a necessity for all these
processes, thus understanding the mechanism of RPA·DNA interaction
may provide insight into the biochemical events that RPA regulates. The
binding of RPA to duplex DNA substrates is not completely understood,
but the affinity is about three orders of magnitude lower compared with
single-stranded DNA, depending on the conditions of the experiment (1,
40). RPA displays preferential binding to duplex damaged DNA compared
with undamaged DNA, and the degree of affinity corresponds with DNA
lesions that are more disruptive to the duplex structure, consistent
with RPA binding and generating single-stranded DNA (4-6). Although
the equilibrium DNA binding activity of RPA has been well characterized (1) and the crystal structure of the RPA·DNA binding domains bound to
an oligo(dC)8 substrate has been solved (37), the kinetics of RPA·DNA binding remain to be elucidated. Here, we have developed a
stopped-flow assay to measure the pre-steady-state kinetics of
RPA-binding DNA. The kinetic data reveals a faster association and
slower dissociation of RPA for duplex cisplatin-damaged DNA compared
with the undamaged DNA, consistent with previous data (5, 6). RPA also
displays differential kinetics for duplex DNA containing a 1,2d(GpG)
cisplatin-DNA adduct compared with the 1,3d(GXG) adduct. RPA associates
about 3-fold faster to the DNA containing the 1,3d(GXG) adduct and
dissociates slower compared with the DNA containing the 1,2d(GpG)
cisplatin-DNA adduct. These data are consistent with the distortion in
the duplex structure generated by the individual adducts and the degree
of single-stranded DNA that RPA can contact. It also correlates with
the difference in the rates of repair seen with the two types of
lesions (22).
The data obtained are consistent with a multistep process for binding
DNA, which involves a rapid association with the duplex DNA and a slow
denaturation step. Following the denaturation, a second molecule of RPA
can associate with the free single-stranded DNA at a rate that is near
diffusion-limited. A recent study measured the equilibrium binding of
RPA with fluorescently labeled damaged and undamaged duplex DNA
substrates using anisotropy (41). The calculated values obtained for
the similar substrates, undamaged duplex, and a duplex containing a
1,3-cisplatin adduct are similar to the values we obtained from rapid
kinetic analysis (Table I). However, the authors argued against the
denaturation of the duplex DNA substrates occurring during the binding
reaction. It is unclear, however, if the analysis performed to come to
this conclusion involved the use of competitor DNA to limit the
re-association of the duplex DNA following digestion with SDS and
proteinase K. We have observed that greater than 80% of a 30-base DNA
can re-anneal with its complement within 1 min after removal from a
95 °C incubation (data not shown). The inclusion of an excess of
unlabeled competitor DNA is essential to accurately measure the
denaturation of short duplex DNAs by electrophoretic mobility shift
assays. Our data, both equilibrium binding and rapid kinetic analysis,
indicate that RPA binds and promotes the dissociation of short duplex
DNA substrates.
The interaction of RPA with single-stranded DNA substrates demonstrated
a slower association and much faster dissociation to single-stranded
DNA containing a single 1,2d(GpG) cisplatin-DNA adduct compared with
the undamaged DNA. This supports our previous model that, upon
recognizing and denaturing the duplex DNA structure around the lesion,
RPA binds to the undamaged strand of the duplex DNA (6). This would
ensure that the undamaged DNA strand is protected from cleavage while
directing the XPG and XPF·ERCC1 proteins to incise 3' and 5' of the
damaged DNA site, respectively. The fast dissociation rate of RPA from
a cisplatin-damaged single-stranded DNA suggests that RPA does not form
a stable interaction with this DNA structure. RPA binding to the
damaged strand would rapidly dissociate then allowing the formation of
a stable interaction with the undamaged strand as a bubble structure
around the adduct is formed. Again, this model for binding is
consistent with our previously published equilibrium binding data using
EMSA analysis (5, 6) and was recently confirmed using fluorescence
anisotropy (41) where a decrease in affinity of RPA for damaged
single-stranded DNA was observed. This data is in contrast to results
reported for the interaction of RPA with single-stranded UV-damaged DNA where RPA is reported to have a much higher affinity for the damaged DNA strand (42). The longer substrates that were used in this study,
however, would not be representative of the actual event that would
occur in DNA damage recognition, in which at best, a small portion of
single-stranded DNA would be exposed for RPA to bind. Using smaller
oligonucleotide substrates, the authors observed only a 2-fold
difference in RPA affinity and suggested that RPA is most likely bound
to both strands during NER due to the minor difference in affinity
between the damaged and undamaged single-stranded DNA (42). This
mechanism seems unlikely due to the strong data supporting RPA binding
polarity (16, 37), the inhibition of incision seen on the strand RPA is
bound (16, 43), and the positioning of the proteins and the known
interactions of RPA with these proteins (14, 18, 21). This also does not rule out the possibility that other protein components like XPA may
influence the positioning of RPA, and if complexed, the potential is
that only one RPA molecule is at the damaged site.
The bimolecular association rate, kon, for
RPA-binding pyrimidine DNA sequences is near diffusion-limited (ranging
from 1.61 to 2.14 nM
1 s
1), and
the difference in affinity observed between DNA length is mainly
attributed to the dissociation rate, koff. These
data support a mechanism of RPA·DNA binding in which more than 15 bases are required to achieve stable DNA binding, and the DNA binding domains within the 70-kDa subunit are not the only portions of the
protein responsible for stable DNA binding. It is possible that the N
terminus, the C terminus, and/or the 34- and 14-kDa subunits play a
role in DNA binding stability. The difference in RPA affinity for
pyrimidine versus purine DNA substrates is manifested mainly
from the kon, whereas the
koff values differ by about 3-fold. Thus, the
DNA sequences influence the association rate at which RPA binds a given
DNA substrate, while having minimal affect on the rate of dissociation.
The association kinetics of cisplatin-DNA damage recognition is
important, because other non-repair proteins have been shown to bind
cisplatin-damaged DNA and have been hypothesized to "shield" the
damaged site from repair (5, 44, 45). The abundant high mobility group
1, HMG1, protein is the prototypical HMG box protein that has been
shown to inhibit NER and helicase catalyzed displacement of
cisplatin-damaged DNA (27, 45). The association rates of the various
proteins for cisplatin-damaged DNA should provide evidence for the
in vivo relevance of the shielding model. Recently, the
kinetics of the HMG1 A and B box binding cisplatin-damaged DNA were
determined, and the data demonstrated a near diffusion-limited binding
to the duplex damaged DNA (30, 46). The association rate of RPA for
duplex cisplatin-damaged DNA is 20 times slower than HMG1 A box,
supporting the shielding model and our previous hypothesis that HMG1
had a faster association to cisplatin-damaged DNA (5). Clearly, the
kinetics of the other repair proteins must be determined, including the
XPC·hHR23B protein, the XPA protein, and the kinetics of the
XPA·RPA complex to support the shielding models in vivo
relevance. It will also be interesting to measure the kinetics of the
wild type HMG1 protein, instead of just the A or B box, to see how the
acidic tail affects the association rate to duplex cisplatin-damaged
DNA, and how the rate of HMG1 association compares to the XPA·RPA complex.
 |
ACKNOWLEDGEMENTS |
We thank Karen Henkels for technical
assistance and Marc Wold for providing the RPA expression vector. We
also thank an anonymous reviewer for helpful comments and suggestions.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Awards CA64374 and CA82741 (to J. J. T.).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.
Supported by a postdoctoral fellowship from Wright State
University Medical School/Biochemistry and Molecular Biology
postdoctoral fellowship program.
§
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Wright State University, 3640 Colonel Glenn
Hwy., Dayton, OH 45435. Tel.: 937-775-2853; Fax: 937-775-3730; E-mail:
john.turchi@wright.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M010314200
 |
ABBREVIATIONS |
The abbreviations used are:
RPA, human
replication protein A;
EMSA, electrophoretic mobility shift assay;
PAGE, polyacrylamide gel electrophoresis;
NER, nucleotide excision
repair;
kon, association rate;
koff, dissociation rate;
XP, xeroderma
pigmentosum;
bp, base pair(s);
HMG1, high mobility group 1;
ssDNA, single-stranded DNA.
 |
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