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J. Biol. Chem., Vol. 276, Issue 21, 18235-18242, May 25, 2001

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Replication Protein A as a "Fidelity Clamp" for DNA Polymerase ^*

Giovanni Maga^§, Isabelle Frouin^¶, Silvio Spadari, and Ulrich Hübscher

From the  Istituto di Genetica Biochimica ed Evoluzionistica-Consiglio Nazionale delle Ricerche, I-27100 Pavia, Italy and the  Institut für Veterinärbiochemie, Universität Zürich-Irchel, 8057 Zürich, Switzerland

Received for publication, October 20, 2000, and in revised form, January 17, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The current view of DNA replication in eukaryotes predicts that DNA polymerase  (pol )-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol  elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol  displays a low processivity and, because of the lack of an intrinsic or associated 3' 5' exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol -primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol  playing a dual role: (i) it stabilizes the pol /primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol . Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol .

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The highly conserved DNA polymerase (pol)¹ -primase complex is the only eukaryotic polymerase able to initiate DNA synthesis de novo, making it of central importance in DNA replication. In fact, it is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork (1-3). The current view of DNA replication in eukaryotes predicts that pol -primase synthesizes the first RNA/DNA primer on the leading strand. Then, at a critical length of 30 nucleotides, replication factor C binds to the 3'-OH end of the nascent DNA strand and displaces pol , thereby loading PCNA and pol . pol -primase switches activity to initiate the synthesis of Okazaki fragments on the lagging strand (4-6). pol -primase has to synthesize short RNA/DNA primers. Thus, its intrinsic low processivity is compatible with its function. However, the switch between primase and polymerase activity, leading to the RNA-to-DNA synthesis transition, occurs through an intramolecular mechanism that does not require dissociation of pol  from the primer (7). Thus, it would be important to ensure stable binding of pol  to the template until completion of the RNA/DNA primer synthesis. In addition, the lack of any intrinsic proofreading function for pol  could lead to misincorporation events during RNA/DNA primer synthesis. The critical roles of the pol -primase make it a likely target for mechanisms that control DNA synthesis initiation and progression (8). In particular, a mechanism ensuring a transient but stable binding of pol  to the primer and increasing its fidelity could represent an advantage for the cell.

The eukaryotic ssDNA-binding protein RP-A is a heterotrimer consisting of three subunits of 70, 32, and 14 kDa (p70, p32, and p14, respectively) (9-11). RP-A has multiple roles in the cell, being essential for DNA replication initiation and elongation (12, 13), DNA repair (14), and DNA recombination (15). These different roles are mediated by direct protein/protein interactions. RP-A has been found to interact physically with the replicative OBP (origin-binding protein)/helicase large T-antigen of SV40 (16-18), with pol -primase (19), with proteins of the nucleotide excision repair (NER) pathway (XPA, XPG, XPF) (20-24), with recombination-specific proteins (15, 25) and with transcriptional activators (GAL4, VP16, p53, RBT1) (26-30). The three subunits of RP-A are likely to play different roles in all these DNA transactions. In particular, mutagenesis studies have begun to reveal different functions of p70, p32, and p14 in DNA replication. All three subunits are required to support DNA replication in vitro. The p70 subunit contains three functionally distinct domains: an N-terminal domain, a central ssDNA-binding domain, and a C-terminal subunit interaction domain (31). The N-terminal domain contains the interaction domain for pol -primase, which consists of two distinct regions: one (amino acids 1-170) that stimulates pol  synthetic activity and another (amino acids 170-327) that increases pol  processivity. This latter region overlaps with the ssDNA-binding domain (amino acids 168-450); such an activity was shown to be required for pol  processivity stimulation by RP-A (19). The p32 subunit interacts with XPA and large T-antigen and is phosphorylated in a cell cycle-dependent manner (32). UV-cross-linking studies with photoreactive primer-templates mapped both p70 and p32 close to the 3'-OH primer end (33-36). The pattern of p70 and p32 labeling was also found to be dependent on the template length and the ratio of RP-A to template concentration. These studies suggested that a series of conformational changes occurs after RP-A binding to ssDNA. Indeed, at least two different modes of RP-A binding to DNA have been detected: one covering 8-10 nucleotides and another with a more extended configuration of 30 nucleotides (37-40). These structurally distinct complexes have been proposed to have different subunits rearrangements.

Very recently, it has further been demonstrated that RP-A, along with the DNA replication protein Cdc45p, is involved in the recruitment of pol -primase at the chromosomal DNA replication origins (41, 42). Thus, both pol -primase and RP-A are simultaneously present and likely to interact physically during initiation of DNA replication. This makes RP-A a likely candidate for the regulation of the catalytic activity of pol -primase.

In the present work, we have investigated the in vitro role of RP-A on the DNA synthetic activity of pol  using different DNA templates. The results obtained indicated that RP-A could assist pol  in two ways: (i) by increasing the stability of the pol/primer complex and (ii) by reducing the overall misincorporation rate of pol .

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals-- [³H]dTTP (40 Ci/mmol) and [-³²P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech; unlabeled dNTPs, poly(dA), and oligo(dT)_12-18, d24-mer, and d66-mer oligodeoxynucleotides were from Roche Molecular Biochemicals. Activated calf thymus DNA was prepared as described (54). Whatman was the supplier of the GF/C and DE-81 filters. All other reagents were of analytical grade and were purchased from Merck or Fluka.

Nucleic Acid Substrates-- The singly primed d24:d66-mer was prepared by labeling the 5'-end of the d24-mer primer with [-³²P]ATP and T4 polynucleotide kinase (Ambion) according to the manufacturer's protocol. The d66-mer template oligonucleotide was then mixed with the complementary labeled d24-mer primer oligonucleotide in a 1:1 molar ratio in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA, heated at 90 °C for 5 min, and then incubated at 65 °C for 2 h and slowly cooled at room temperature.

The sequence of the d66-mer oligonucleotide was: 5'-AGGATGTATGTTTAGTAGGTACATAACTATCTATTGATACAGACCTAAAACAAAAAATTTTCCGAG-3'. The sequence of the d24-mer oligonucleotide was: 5'-CTCGGAAAATTTTTTGTTTTAGGT-3'.

Enzymes and Proteins-- Calf thymus pol  was purified as described (54). The pol  used in this study was 250 units/ml (0.2 mg/ml). 1 unit of pol activity corresponds to the incorporation of 1 nmol of total dTMP into acid-precipitable material for 60 min at 37 °C in a standard assay containing 0.5 µg (nucleotides) of poly(dA)/oligo(dT)_10:1 and 20 µM dTTP. Recombinant human RP-A was isolated as described (55).

Enzymatic Assays-- pol  activity on poly(dA)/oligo(dT)_10:1 was assayed in a final volume of 25 µl containing 50 mM Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mM dithiothreitol, 6 mM MgCl_2, and 5 µM [³H]dTTP (5 Ci/mmol), unless otherwise indicated in the figure legends. All reactions were incubated for 15 min at 37 °C unless otherwise stated, and the DNA was precipitated with 10% trichloroacetic acid. Insoluble radioactive material was determined as described (56). When the singly primed d24:d66-mer oligodeoxynucleotide was used as template, a final volume of 25 µl contained 50 mM Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mM dithiothreitol, 6 mM MgCl_2, and 10 µM each [³H]dATP (5 Ci/mmol), dGTP, dCTP, and [³H]dTTP (5 Ci/mmol). Enzymes and proteins were added as indicated in the figure legends. All reactions were incubated for 15 min at 37 °C unless otherwise stated and stopped by the addition of 0.1 M EDTA and 1 µg of calf thymus DNA as carrier. 20 µl of the reaction mixture were then spotted onto DE-81 cellulose filters. Filters were washed to remove unincorporated dNTPs as described (57), and incorporated radioactivity was monitored by scintillation counting.

For incorporation studies with the singly primed d24:d66-mer oligodeoxynucleotide as template, a final volume of 10 µl contained 50 mM Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mM dithiothreitol, 6 mM MgCl₂, and 20 nM (3'-OH ends) of the 5' ³²P-labeled d24:d66-mer DNA template. Enzymes, proteins, and unlabeled dNTPs were added as indicated in the figure legends. All reactions were incubated for 15 min at 37 °C, samples were mixed with denaturing gel loading buffer (95% v/v formamide, 10 mM EDTA, 0.25 mg/ml bromphenol blue, 0.25 mg/ml xylene cyanol), heated at 95 °C for 5 min, and then subjected to electrophoresis on a 7 M urea, 20% polyacrylamide gel. Quantification of the reaction products on the gel was performed using a Molecular Dynamics PhosphorImager and ImageQuant software.

Steady-state Kinetic Data Analysis-- K_m, V_max, and [RP-A]₅₀ values were calculated according to the Michaelis-Menten equation in the form,

(Eq. 1)

where k_catE₀ = V_max. The K_i values for incorrect dNTPs were determined from inhibition assays with increasing concentrations of the selected dNTP in the presence of different fixed amounts of RP-A and were calculated according to a simple competitive mechanism of inhibition as described by the equation,

(Eq. 2)

Computer fitting of the experimental data to the equations was performed with the program MacCurveFit^TM 1.5 using the least squares curve-fitting quasi-Newton method, based on the Davidon-Fletcher-Powell algorithm (59).

When data points were derived from densitometric analysis of the intensities of the products bands, the values of integrated gel band intensities in dependence of the nucleotide substrate concentrations were fitted to the equation (58),

(Eq. 3)

where, T = target site, the template position of interest; and I*T = the sum of the integrated intensities at positions T, T+1 ... T+n.

Before being inserted in the above equation, the intensities of the single bands of interest were first normalized by dividing for the total intensity of the lane. This was done to reduce the variability because of manual gel loading. An empty portion of the gel was scanned, and the resulting value was subtracted as background. The goodness of the interpolated curve was assessed by computer-aided calculation of the sum of squares of errors and the correlation coefficient R². Standard errors were provided by the computer program MacCurveFit^TM 1.5. The standard errors are calculated from the variance-covariance matrix, and the values displayed are the square roots of the diagonal elements. The variance-covariance matrix is calculated from the Jacobian matrix (59).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

RP-A Stimulates the Synthetic Activity of pol  in Dependence of the 3'-OH Primer Concentration-- Different concentrations of RP-A were tested for their effect on nucleotide incorporation catalyzed by pol  on different DNA templates (Fig. 1). RP-A was able to stimulate pol  activity on either homo- or heteropolymeric deoxyoligonucleotides, within a range of concentrations close to the 3'-OH primer concentration used in the assay (Fig. 1A). Variation of the 3'-OH primer concentration resulted in a shift of the RP-A concentration giving the maximal stimulation, which was observed at equimolar amounts of RP-A to 3'-OH primer (Fig. 1B, arrows).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   RP-A stimulates the synthetic activity of pol  in dependence of the 3'-OH primer concentration. Reactions were performed as described under "Materials and Methods" with 0.012 units of pol . A, DNA synthesis by pol  was measured in the presence of increasing amounts of RP-A (as trimer) with 60 nM (3'-OH ends) of either the homopolymeric substrate poly(dA)/oligo(dT)12-18 (circles) or the heteropolymeric oligodeoxynucleotide d24:d66-mer (squares). B, DNA synthesis by pol  was measured in the presence of increasing amounts of RP-A (as trimer) in the presence of 25 nM (3'-OH ends) (squares), 75 nM (3'-OH ends) (triangles), or 125 nM (3'-OH ends) (circles) of poly(dA)/oligo(dT)12-18. The arrows indicate maximal stimulation at equimolar RP-A to 3'-OH primer concentrations.

RP-A Increases the Affinity of pol  for 3'-OH Primers-- To investigate the effects of RP-A on the nucleotide incorporation reaction catalyzed by pol , increasing concentrations of 3'-OH primer were tested in the presence of different fixed amounts of RP-A. As shown in Fig. 2A, the K_m value of pol  for the 3'-OH primer was decreased by increasing concentrations of RP-A. The V_max/K_m ratio, which is an estimate of the association rate for the pol ·3'-OH primer complex formation, was also increased by RP-A (Fig. 2B). A comparison of the variation of the reaction velocity in dependence of the 3'-OH primer concentration (Fig. 2C) with the observed decrease of K_m values in dependence of RP-A concentration (Fig. 2D) showed that [RP-A]₅₀, the RP-A concentration giving half of the maximal decrease, was 41 nM, a value very close to the K_m of pol  for the 3'-OH primer, which is 39 nM.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   RP-A increases the affinity and the association rate of pol  for 3'-OH primers. A, Km values were calculated for different combinations of DNA and RP-A and values plotted in dependence of the RP-A concentrations. 0.012 units of pol  were tested as described under "Materials and Methods" in the presence of 12.5, 25, 50, 125, and 250 nM (3'-OH ends) poly(dA)/oligo(dT)12-18 and in the presence of increasing amounts of RP-A (as trimers). B, plot of the ratio between the Vmax and Km values derived from the experiments described for panel A in dependence of the RP-A concentration. C, dependence of pol  synthesis on DNA concentration in the absence of RP-A. 0.012 units of pol  were tested in the presence of 12.5 nM, 25, 50, 125, and 250 nM (3'-OH ends) poly(dA)/oligo(dT)12-18. Fitting of the curve to the Michaelis-Menten equation was performed by computer simulation. Inset, time course of the reaction in the presence of 300 nM (3'-OH ends) poly(dA)/oligo(dT)12-18. The reaction was linear within the first 20 min of incubation. The maximal incorporation observed corresponded to 5% of the total dTTP substrate concentration, thus indicating that the measured rate was the true initial velocity of the reaction. D, the difference between the affinity constant for the 3'-OH primer calculated in the absence (Km) or in the presence (Kapp) of increasing amounts of RP-A, as shown in panel A, was calculated, and the resulting values ( = Km  Kapp) were plotted in dependence of the RP-A concentration. Fitting to the equation  = max/(1 + ([RP-A]50/[RP-A])) was performed by computer simulation.

RP-A Increases the Ability of pol  to Discriminate between Correct and Incorrect Nucleotide Incorporation-- Next, the effect of RP-A on the ability of pol  to incorporate a wrong nucleotide was tested. It is known that the misincorporation efficiency of a pol is influenced by the nature of the mismatch resulting from the misalignment of the template-encoded base and the incoming nucleotide (43). Thus, to directly compare the effects of RP-A on the ability of pol  to incorporate a wrong nucleotide, the homopolymeric substrate poly(dA)/oligo(dT) was used, which contains only adenines as template. As shown in Fig. 3A, the incorporation of radioactively labeled dTTP catalyzed by pol  on such a template can be inhibited by the addition of unlabeled dNTPs as competitors. Each individual dNTP inhibited the reaction with different potencies, as indicated by the different K_i values (Table I). The calculated K_i values and the K_i/K_m ratios, which are an estimate of the accuracy of nucleotide incorporation by pol , are listed in Table I. pol  discriminates 330-fold against the C-A mismatch, but only 60-fold against the A-A and 30-fold against the G-A misincorporations, respectively. Thus, pol  showed different misincorporation efficiencies for the resulting mismatches, in the order G-A > (i.e. more efficiently generated) A-A > C-A. When similar experiments were performed in the presence of RP-A, the misincorporation efficiencies for the G-A and A-A mismatches were reduced to about the level observed for the C-A misincorporation (see below and Table I).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   RP-A increases the ability of pol  to discriminate between correct and incorrect nucleotide incorporation. A, 0.012 units of pol  were tested as described under "Materials and Methods" in the presence of 250 nM (3'-0H ends) poly(dA)/oligo(dT)12-18 and 2 µM [3H]dTTP and in the absence or presence of increasing amounts of unlabeled dTTP (triangles), dCTP (circles), dGTP (squares), or dATP (rhomboids). pol  activity expressed as percent of the control without added dNTPs was plotted in dependence of the dNTPs concentration. B, reactions were performed as described for panel A but in the presence of increasing amounts of dGTP only and in the absence (triangles) or presence of 50 (circles), 120 (rhomboids), or 250 nM (squares) RP-A (as trimer). pol  activity expressed as percent of the control without added dGTP was plotted in dependence of the dGTP concentration. C, the increase in the inhibition constant (-fold Ki) for dGTP was calculated as the ratio among the Ki values for dGTP in the absence of RP-A and the corresponding values in the presence of RP-A, plotted in dependence of the RP-A concentration. Fitting to the equation (-fold Ki) = (-fold Ki)max/(1 + ([RP-A]50/[RP-A])) was performed by computer simulation.

View this table: [in this window] [in a new window]   Table I Effect of RP-A on the inhibition by different dNTPs of dTTP incorporation catalyzed by pol  on poly(dA)/oligo(dT)

RP-A Decreases the Misincorporation Efficiency of pol  in a Concentration-dependent Manner-- Fig. 3B shows the effect of different amounts of RP-A on the inhibition by dGTP of dTTP incorporation catalyzed by pol  on poly(dA)/oligo(dT). The plot of the increase in the K_i value of dGTP versus RP-A concentration showed a typical saturation kinetics (Fig. 3C), with a half-maximal stimulatory RP-A concentration (or [RP-A]₅₀) of 47 nM, very close to the value observed for the 3'-OH primer binding stimulation (Fig. 2D). Maximal increase of the K_i values for dGTP inhibition was observed at RP-A concentrations close to the concentration of 3'-OH. Similar results were obtained when dATP inhibition was tested (data not shown).

Sequencing Gel Analysis of the Products of Nucleotide Incorporation Catalyzed by pol  on the d24:d66-mer Template-- Because the template sequence was known, it was possible to force pol  to make specific mismatches by adding the appropriate combinations of nucleotides in a reaction mixture. Fig. 4 shows the products of a reaction containing the heteropolymeric deoxyoligonucleotide resolved by denaturing polyacrylamide gel electrophoresis. The addition of different combinations of nucleotides generated strong pausing sites at the positions immediately preceding the mismatch, suggesting that incorporation of a wrong nucleotide was rate-limiting with respect to the elongation of a mismatched primer. For example, in the presence of the first encoded nucleotide, dCTP, a strong signal was detected at position +1 (lane 2), whereas the addition of the first two nucleotides, dCTP and dTTP, resulted in the generation of a strong band at position +2 (lane 3). Significant misincorporation products were detected with all of the combinations used (lanes 2-4), confirming the relatively low fidelity of pol  in DNA synthesis.

View larger version (103K): [in this window] [in a new window]   Fig. 4.   Sequencing gel analysis of the products of nucleotide incorporation catalyzed by pol  on the d24:d66-mer template. Reactions were performed as described under "Materials and Methods" in the presence of 0.02 units of pol  and different combinations of dNTPs, as indicated at the bottom of the figure, in the absence (lanes 1-4) or presence (lanes 5-8) of 25 nM RP-A as trimer. Lane 9, 5'-labeled 24-mer oligonucleotide primer.

RP-A Decreases the Amount of Misincorporated Products Synthesized by pol -- The same reactions were performed in the presence of RP-A (Fig. 4, lanes 5-8). The first observation was that RP-A stimulated the correct incorporation of nucleotides by pol  as judged by the increasing intensities of the bands corresponding to complementary nucleotide incorporation (Fig. 4, compare positions 0, +1, and +2 in lanes 2-4 with the same positions in lanes 5-8). In particular, the band at position 0 decreased significantly; this was expected, because RP-A imposed a block to misincorporation, which forced pol  to elongate mainly the d24-mer primer to a d25-mer product but did not allow further elongation because of the lack of the complementary correct nucleotides required. The intensities of the bands were quantified by densitometric analysis, and the relative amounts of synthesized products at each position along the template were calculated as described in the figure legend, both in the absence and in the presence of RP-A. As shown in Fig. 5, with the different combinations of nucleotides tested, RP-A significantly decreased the amount of products generated by elongation of a mismatched primer by pol , thereby increasing the accumulation of products at the position immediately preceding the misincorporation site (compare panel A with B in Fig. 5).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   RP-A decreases nucleotide misincorporation catalyzed by pol . The products of incorporation reactions performed as described for Fig. 4 were resolved by sequencing gel electrophoresis. The amounts of product synthesized at each position along the template were determined by densitometric analysis of the corresponding bands and expressed as relative values (% of Itot, where Itot is the sum of the intensities of all the bands in the same lane) to account for small differences arising from manual gel loading. A, products synthesized by pol  in the presence of 50 µM dCTP (light gray bars) or a combination of 50 µM dCTP and 50 µM dTTP (dark gray bars). Template positions along with the template sequence (one-letter codes in brackets) are indicated. B, same as in panel A but in the presence of 25 nM RP-A. Error bars indicate the errors (±S.D.) calculated over three independent experiments. The significance of the differences between the mean values obtained from reactions with and without RP-A were tested by a Student's t test under the null hypothesis that the true mean values were equal in all cases. The probability value that the reference hypothesis was true was p  0.05, and thus the observed differences were considered statistically significant.

RP-A Influences Both the K_m and the V_max of Incorrect but Not of Correct Nucleotide Incorporation Catalyzed by pol -- To investigate more closely the mechanism by which RP-A decreases the misincorporation efficiency of pol , a detailed kinetic analysis of correct versus incorrect single nucleotide incorporation was performed. Reactions were carried out as shown in Fig. 4, lanes 2 and 6, respectively, but in the presence of different concentrations of dCTP. Quantification of the products at position +1 (C-G base pair) and +2 (C-A mismatch) at each nucleotide concentration allowed the calculation of the K_m and V_max values for the correct and incorrect incorporation reactions, as well as the specificity constant V_max/K_m. The computed values are listed in Table II. RP-A specifically decreased both the affinity and the reaction velocity for the misincorporation reactions, whereas it only slightly stimulated the correct nucleotide incorporation (~2-fold increase in the V_max/K_m value). A comparison of the ratio between the V_max and the K_m values for correct and incorrect nucleotide incorporation in the absence and the presence of RP-A showed that pol  alone discriminated ~540-fold against the C-A mismatch, a value comparable with the one derived with poly(dA)/oligo(dT) (Table I). This value was increased to more than 3300-fold by the addition of RP-A, thus reducing the misincorporation more than 6-fold (Table II).

View this table: [in this window] [in a new window]   Table II Effect of RP-A on the kinetic parameters for correct (C-G) versus incorrect (C-A) single nucleotide incorporation by pol

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characterization of the Functional Interaction between RP-A and pol -Primase-- Several lines of evidence suggested that RP-A makes close contacts with both pol  and the 3'-OH primer end. UV-cross-linking experiments showed the p70 subunit of RP-A bound to the single-stranded DNA of a primer-template junction, whereas the p32 subunit was cross-linked very close to the 3'-OH primer end (33-36). It has been shown by mutagenesis experiments that pol  binds to the p70 and p32 subunits of RP-A. In particular, it was shown that the N-terminal half of the DNA-binding domain of the p70 subunit (amino acids 170-327) was responsible for the enhancement of pol  processivity and that the ssDNA binding activity of RP-A was required to achieve this stimulation (19). To investigate the molecular mechanisms and the functional significance of the RP-A/pol  interaction, we examined in detail the effect of RP-A on pol  catalytic activity. Our results clearly showed that RP-A was able to stimulate pol  activity on different DNA templates (Fig. 1A). Under the assay conditions used, maximal stimulation was always achieved with an RP-A concentration equal to the 3'-OH primer ends concentration (Fig. 1B). When the effect of RP-A on the DNA binding affinity of pol  was studied, the results showed that RP-A could increase the primer binding activity of pol  (Fig. 2, A and B). Interestingly, the amount of RP-A required for half-maximal stimulation was almost identical to the concentration of 3'-OH primer ends required to half-saturate the pol  active site (Fig. 2, C and D). These results might suggest that binding of RP-A leads to the formation of a ternary pol ·3'-OH primer·RP-A complex, with 1:1:1 stoichiometry and with increased stability with respect to the binary pol ·3'-OH primer complex. Stimulation of primer binding affinity was observed only in the presence of stoichiometric amounts of RP-A to the 3'-OH primer. An excess of RP-A suppressed this effect. This observation is consistent with the results of Braun et al. (19), who showed that processivity stimulation of pol  by RP-A was restricted to subsaturating amounts of RP-A. Thus, the stoichiometry of RP-A binding to the pol ·3'-OH primer complex is crucial for its function. UV-cross-linking studies with RP-A and photoreactive primer templates showed that the labeling pattern of p70 and p32, and consequently their interaction with the template-primer junction, was strongly dependent on the ratio of RP-A to template concentration (36). A conformational change, involving repositioning of the p32 and p70 subunits, has been proposed to occur when saturating amounts of RP-A molecules associate during cooperative DNA binding. It is possible that, under such conformation, RP-A can no longer stabilize the pol ·3'-OH primer complex. Thus, transiently limiting RP-A binding to stoichiometric amounts with respect to the available template-primer junctions in the presence of pol  might represent a novel mechanism for regulation of the early events in DNA synthesis. RP-A was not found to influence the polymerization rate of pol , but the observed increase in primer binding stability could nevertheless account for the increase in pol  processivity described by Braun et al. (19). A major difference among pol  and the other two replicative pols, pol  and pol , is the lack of an intrinsic (or functionally associated) 3' 5' proofreading exonuclease (3). As a result, the overall misincorporation rate of pol  has been estimated at about 2.5 × 10⁴ (44), approximately 5-20-fold higher than for pol  and pol . The RP-A-dependent change in the molecular structure of the pol ·3'-OH primer complex could potentially influence the ability of pol  to tolerate a misaligned nucleotide within its active site (45, 46). Indeed, the fidelity of DNA synthesis catalyzed by pol  when part of a multiprotein replication complex was found to be higher than that of purified pol , suggesting a role for accessory factors (47, 48). Using a combination of in vitro DNA synthesis and genetic screening for detecting mutations in Escherichia coli, RP-A has been found to increase by 5-6-fold the fidelity of pol , even if no explanation for this effect at the molecular level was presented (49). However, another study with a different approach failed to detect a specific role of RP-A in modulating pol  fidelity (50). In the present study, when the effect of RP-A on the ability of pol  to incorporate wrong nucleotides was analyzed in in vitro assays with purified proteins, the results showed that the overall misincorporation efficiency was reduced by about 5-6-fold (Figs. 3 and 5 and Tables I and II). A kinetic analysis showed that this effect was driven by RP-A physical association with pol  and the 3'-OH primer end, which reduced both the affinity and the catalytic efficiency of pol  for incorrect versus correct nucleotide incorporation (Table II). Thus, the ternary pol ·3' OH primer·RP-A complex, besides having an increased stability, also showed a reduced ability to tolerate misalignments between the DNA template and the incoming nucleotide.

RP-A as a Fidelity Clamp-- The role of the auxiliary proteins in DNA replication is to provide pols with particular properties, which make them better suited to perform the difficult task of replicating the whole cellular genome (9). For example, highly processive DNA synthesis is achieved by replicative pols through their interaction with a class of auxiliary proteins called sliding clamps or processivity clamps. In eukaryotic cells, such a role is fulfilled by the accessory protein PCNA, which acts as a processivity factor for both pol  and pol , increasing their primer binding efficiency. The presence of a strongly evolutionary conserved processivity factor for pol  is consistent with its role in the DNA replication process, namely elongation of leading and lagging strands. On the other hand, both pol  and pol  possess a 3' 5' proofreading exonuclease activity, and thus they should not require any fidelity-enhancing factor. pol  performs a different task in DNA replication, synthesizing the short (30 nucleotides) RNA/DNA primers required for subsequent processive DNA synthesis by pol  (or pol ) (4). The switch between primase and pol activity, leading to the RNA-to-DNA synthesis transition, occurs through an intramolecular mechanism that does not require dissociation of pol  from the primer (7). Additionally, the lack of any intrinsic proofreading function for pol  could lead to misincorporation events during primer synthesis (44). The results presented here indicate that the RP-A-template-primer complex is a better substrate for pol , allowing more processive and accurate synthesis of the RNA/DNA primers. Under this respect, RP-A behaves differently from the sliding clamps PCNA and gp45, which have been shown to reduce the accuracy of the cognate pols, allowing these enzymes to more easily replicate across DNA lesions and to extend mispaired primer ends (51, 52). Thus, RP-A might represent the first example of a novel class of auxiliary factors, which we propose to call "fidelity clamps." Recent data suggested that a preinitiation complex between RP-A, Cdc45p, Mcm4p, and pol  is formed at the replication origins (41, 42). Moreover, pol /RP-A interaction is also important in the switch between pol  and pol  at the lagging strand, which is dependent on replication factor C and PCNA (5, 53). Thus, it appears that a continuous interaction between RP-A and pol -primase is maintained throughout the early steps of DNA replication.

	FOOTNOTES

^* This work was supported by grants from the European Union-Training and Mobility of Human Resources program (EU-TMR) (ERBMRXCT 970125 to S. S. and U. H.), the Consiglio Nazionale delle Ricerche Target Project on Biotechnology (to S. S.), and the Kanton of Zürich (to U. H. and G. M.).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 fellowship from the EU-TMR Network (Grant ERBMRXCT 970125).

^§ To whom correspondence should be addressed: IGBE-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy. Tel.: 39-0382546355; Fax: 39-0382422286; E-mail: maga@igbe.pv.cnr.it.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009599200

	ABBREVIATIONS

The abbreviations used are: pol //, DNA polymerases //; PCNA, proliferating cell nuclear antigen; RP-A, replication protein A; ss, single-stranded.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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