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. PatrickDagger 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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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, 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-beta -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 iff  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).

    RESULTS
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INTRODUCTION
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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.

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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Table I
Kinetic parameters of RPA-binding DNA

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.
<UP>RPA</UP>+<UP>dsDNA</UP> <LIM><OP><ARROW>⇔</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>RPA</UP> · <UP>dsDNA</UP> <LIM><OP><ARROW>⇔</ARROW></OP><LL><UP>k<SUB>−s</SUB></UP></LL><UL>k<SUB><UP>s</UP></SUB></UL></LIM><UP> RPA</UP> · <UP>dsDNA*</UP>

<UP><SC>Reaction</SC> 1</UP>
This model predicts a hyperbolic curve of kobs versus DNA concentration and the data were fit to Eq. 1 (35).
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>−s</UP></SUB>+<FENCE><FR><NU>k<SUB><UP>s</UP></SUB>[<UP>DNA</UP>]</NU><DE>K<SUB>D</SUB>+[<UP>DNA</UP>]</DE></FR></FENCE> (Eq. 1)
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.
<UP>RPA</UP>+<UP>dsDNA </UP><LIM><OP><ARROW>⇔</ARROW></OP><LL><UP>k<SUB>−1</SUB></UP></LL><UL><UP>k<SUB>1</SUB></UP></UL></LIM><UP> RPA</UP> · <UP>dsDNA</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM><UP> RPA</UP> · <UP>ssDNA</UP>+<UP>ssDNA</UP>

<UP>ssDNA</UP>+<UP>RPA </UP><LIM><OP><ARROW>⇔</ARROW></OP><LL><UP>k<SUB>−3</SUB></UP></LL><UL><UP>k<SUB>3</SUB></UP></UL></LIM><UP> RPA</UP> · <UP>ssDNA</UP>

<UP><SC>Reaction</SC> 2</UP>
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.

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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha /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.

Dagger 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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RESULTS
DISCUSSION
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