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INTRODUCTION |
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 |
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
MgCl2, 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 MgCl2, 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--
Km, Vmax,
and [RP-A]50 values were calculated according to
the Michaelis-Menten equation in the form,
|
(Eq. 1)
|
where kcatE0 = Vmax. The Ki 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 MacCurveFitTM 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 MacCurveFitTM 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 |
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).

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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.
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|
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
Km value of pol
for the 3'-OH primer was
decreased by increasing concentrations of RP-A. The
Vmax/Km 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 Km 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 Km of pol
for the 3'-OH primer, which is 39 nM.

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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.
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|
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 Ki values
(Table I). The calculated
Ki values and the
Ki/Km 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).

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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.
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Table I
Effect of RP-A on the inhibition by different dNTPs of dTTP
incorporation catalyzed by pol on poly(dA)/oligo(dT)
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|
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 Ki 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 Ki
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.

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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.
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|
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).

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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.
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RP-A Influences Both the Km and the Vmax 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
Km and Vmax values for the
correct and incorrect incorporation reactions, as well as the
specificity constant Vmax/Km.
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
Vmax/Km value). A comparison of the ratio between the Vmax and the
Km 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).
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Table II
Effect of RP-A on the kinetic parameters for correct (C-G) versus
incorrect (C-A) single nucleotide incorporation by pol
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|
 |
DISCUSSION |
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 × 10
4 (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.