From the
Human replication protein A (RPA) is a three subunit protein
complex involved in DNA replication, repair, and recombination. We
investigated the role of the 34-kDa subunit (p34) of RPA in DNA
replication by generating a series of p34 mutants. While deletion of
the N-terminal domain of p34 prevented its phosphorylation by both
cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point
mutant that lacks the major phosphorylation sites for Cdk could be
phosphorylated by DNA-dependent kinase. In simian virus 40
(SV40) DNA replication, RPA containing either of these mutants
functioned as efficiently as wild-type RPA. However, mutant RPA
containing C-terminally deleted p34 was only marginally active. This
indicates that the C-terminal region, but not the phosphorylation
domain of p34, is necessary for RPA function in DNA replication.
Furthermore, RPA containing the C-terminally deleted p34 mutant could
stimulate DNA polymerase
The in vitro simian virus 40 (SV40)
The cDNAs encoding the human RPA
subunits are about 30% homologous to their yeast counterparts (Erdile
et al., 1990, 1991; Heyer et al., 1990; Brill and
Stillman, 1991; Umbricht et al., 1993). However, yeast RPA
substitutes poorly for human RPA in the in vitro SV40
replication system (Brill and Stillman, 1989), indicating that highly
specific protein-protein interactions occur between RPA and other
replication protein(s). SV40 T Ag, RPA, and the DNA polymerase
RPA p34 is phosphorylated on serine residues at the G
Recent evidence from
Cdk-depleted S-phase extracts suggests that RPA can associate with both
single-stranded DNA and SV40 origin-containing DNA, and then become
phosphorylated even in the absence of S-phase kinases (Fotedar and
Roberts, 1992). It may be that the cell cycle-dependent phosphorylation
of RPA p34 results from the coordinated action of Cdk and DNA-dependent
kinase both of which function at the G
In this report, we address the role of RPA p34 in DNA
replication by comparing the function of wild-type RPA with that of a
series of mutants. Deletion of the N-terminal region of RPA p34
abolished its phosphorylation by both Cdk and DNA-dependent kinase but
did not affect the mutant's ability to support SV40 DNA
replication in vitro. A double point mutant that lacks Cdk
phosphorylation sites also retains its activity in the SV40 replication
system. However, mutant RPA lacking the C-terminal region of p34 only
weakly supported DNA replication and unwinding, and interacted poorly
with SV40 T Ag. We discuss the implications of these results on the
role of RPA p34 in DNA replication.
Replication products were analyzed using
[
We, therefore,
examined whether our RPA p34 mutants supported SV40 DNA replication
in vitro. RPA containing p34
Previously, the p70 subunit of RPA alone was shown to be required by
pol
RPA has been implicated in the regulation of DNA replication
and repair. Its three subunit structure is conserved among the
eukaryotes, suggesting a specific role for each subunit. However, no
specific function for either the p34 or the p11 subunit has yet been
identified.
To investigate the role of the RPA p34 subunit, we
created a set of deletion/substitution mutants. Two deletion mutants
were particularly interesting: p34
It has been suggested
that RPA phosphorylation by Cdk is involved in the activation of
G
DNA-dependent kinase phosphorylates RPA p34 only after RPA
associates with ssDNA (Fotedar and Roberts, 1992; Pan et al.,
1994; this work). Immunofluorescence studies indicate that RPA p34 and
Cdk2-cyclin A colocalize to the replication foci during S-phase
(Cardoso et al., 1993). These results suggest that, in
vivo, RPA p34 phosphorylation may occur only after RPA has been
recruited to the replication origin where it may be involved in the
regulation of DNA synthesis. Furthermore, UV irradiation and ionizing
radiation of human cells have been shown to induce hyperphosphorylation
of RPA p34 (Liu and Weaver, 1993; Carty et al., 1994) and to
reduce the ability of RPA to support in vitro DNA replication
(Carty et al., 1994). This suggests that RPA phosphorylation
may be involved in the regulation of DNA replication induced by DNA
damage.
Deletion of 33 amino acids at the C terminus of RPA p34
significantly reduced the level of SV40 DNA replication activity in the
presence of crude extracts, indicating that this region of p34 is
necessary for RPA function in DNA replication. Our data suggest that
the C-terminal region of RPA p34 is involved in the interaction between
RPA and SV40 T Ag, an interaction presumed to occur during the
initiation stage of DNA replication. The unwinding activity of mutant
RPA was considerably reduced compared to that of wild-type RPA,
suggesting that the interaction between RPA and SV40 T Ag is essential
for efficient unwinding of SV40 origin-containing DNA. It should be
pointed out, however, that the ssDNA binding activity of RPA containing
p34
The
anti-p34 monoclonal antibody inhibits both in vitro SV40 DNA
replication and the stimulatory effect of RPA on pol
We thank C. J. Sherr for providing recombinant
baculovirus clones encoding cyclin A and Cdk2, R. Drissi for scientific
discussion and critical reading of the manuscript, E. Stigger for help
throughout the studies, T. Weeden for excellent technical assistance,
and S. Vallance for editing the manuscript. We also thank the St. Jude
Molecular Resource Center for DNA sequencing and oligonucleotide
synthesis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, and bind to single-stranded DNAs but
was limited in its ability to unwind DNA or interact with SV40 large T
antigen (T Ag). These results suggest that RPA p34 interacts with SV40
T Ag during the initiation of SV40 DNA replication and may be necessary
for DNA unwinding.
(
)
DNA replication system has been used extensively as a model
to understand eukaryotic DNA replication because it uses the host
replication machinery for its own DNA replication together with the
virally encoded SV40 large T antigen (T Ag). The development of
cell-free SV40 DNA replication (Li and Kelly, 1984; Wobbe et
al., 1985; Stillman and Gluzman, 1985) has led to the
identification of a number of human replication factors involved in
SV40 DNA replication in vitro (Challberg and Kelly, 1989;
Stillman, 1989; Hurwitz et al., 1990) including human
replication protein A (RPA, also called human single-stranded
DNA-binding protein or HSSB) (Wobbe et al., 1987; Fairman and
Stillman, 1988; Wold and Kelly, 1988). Human RPA comprises three
subunits of 70, 34, and 11 kDa (p70, p34, and p11, respectively), which
are tightly associated with each other (Fairman and Stillman, 1988) and
are conserved among other species (Brill and Stillman, 1989, 1991;
Mitsis et al., 1993; Brown et al., 1993). RPA
subunits are assembled in an ordered process; p34 forms a stable
complex with p11 to which p70 binds (Stigger et al., 1994;
Henricksen et al., 1994).
-primase complex (pol
-primase) functionally interact in
vitro to form a primosome complex that is required for both primer
synthesis (Collins and Kelly, 1991; Melendy and Stillman, 1993) and DNA
synthesis at the replication fork (Murakami and Hurwitz, 1993).
Physical interactions between the various components of the primosome
have been shown by enzyme-linked immunosorbent assay (ELISA) and
immunoblotting (Dornreiter et al., 1992; Collins et
al., 1993). RPA functions at the initiation stage of SV40 DNA
replication by promoting both T Ag-dependent presynthetic unwinding of
SV40 DNA (Dean et al., 1987; Wold et al., 1987;
Borowiec et al., 1990) and pol
-primase activity
(Matsumoto et al., 1990). During the elongation stage, RPA
stimulates the activities of pol
-primase, pol
, and pol
(Kenny et al., 1989; Lee et al., 1991). While
RPAs from Escherichia coli (SSB), adenovirus (DNA-binding
protein (Ad-DBP)), and herpesvirus (ICP8) can substitute for human RPA
to stimulate T Ag-catalyzed unwinding of SV40 DNA and pol
activity in the SV40 system, they cannot replace human RPA to stimulate
pol
-primase activity (Kenny et al., 1989). This suggests
that the interaction between RPA and pol
-primase may contribute
to the species specificity of SV40 replication. The roles of the
individual RPA subunits, particularly p34 and p11, in DNA metabolism
are not known. In yeast, all three RPA genes, rpa1,
rpa2, and rpa3 (the RPA p70, p34, and p11 homologs,
respectively), are essential for cell viability (Brill and Stillman,
1991). Although in yeast, RPA is not required for cell cycle entry,
disruption of the rpa genes results in an abnormal phenotype
that is consistent with an S-phase defect (Brill and Stillman, 1991).
/S
transition and then dephosphorylated during mitosis (Din et
al., 1990; Dutta et al., 1991; Dutta and Stillman, 1992).
One of the kinases responsible for p34 phosphorylation is a
cyclin-dependent kinase (Cdk) (Elledge et al. 1992; Dutta and
Stillman, 1992; Pan et al., 1993; Pan and Hurwitz, 1993).
Several reports implicate this kinase in DNA replication. First, in
SV40 DNA unwinding experiments with T Ag and HeLa cell cytosolic
extracts, G
-phase cell extracts have a much lower DNA
unwinding activity than S-phase or G
-phase extracts.
However, G
-phase cell extracts could be activated by the
addition of Cdk (Roberts and D'Urso, 1988; D'Urso et
al., 1990). Second, depletion of Cdk from Xenopus egg
extracts results in the inability of those extracts to support DNA
replication (Fang and Newport, 1991; Blow and Nurse, 1990). To date,
there is no direct evidence that the activation of DNA replication is
due to RPA p34 phosphorylation by Cdk.
/S transition. RPA
p34 is also phosphorylated following DNA damage caused by UV (Carty
et al., 1994) or ionizing radiation (Liu and Weaver, 1993).
However, the role of RPA p34 phosphorylation in DNA metabolism remains
unknown.
Cell Extracts, Proteins, Antibodies, and
DNA
HeLa cell cytosolic extracts and their ammonium sulfate
fractions were prepared as described previously (Wobbe et al.,
1985, 1987), as was SV40 T Ag (Lee et al., 1991). Pol
-primase and topoisomerase I (topo I) were isolated from HeLa
cells using the procedure described by Ishimi et al.(1988).
Monoclonal antibodies against RPA p70 and p34 were generated as
described earlier (Kenny et al., 1990), and DNA primers for
PCR, oligo(dT)
, and oligo(dT)
were
synthesized by the Molecular Resource Center at St Jude
Children's Research Hospital, Memphis, TN.
Recombinant Baculoviruses
Recombinant baculovirus
constructs encoding wild-type p70, p34, and p11 have been described
previously (Stigger et al., 1994). The double point mutant, in
which alanine is substituted for serine at amino acids 23 and 29 in p34
(p34(S/A:23,29)), was generated by polymerase chain reaction (PCR)
using the full-length cDNA and the following sets of primers: 5`-CGC
GGA TCC ATG TGG AAC-3` and 5`-GCG CTC CAA AGC CCC CCG GGG ACC GCG-3`
(representing the N-terminal sequences and amino acids 23-29),
and 5`-CGC GGT CCC CGG GGG GCT TTG GAG CGC-3` and 5`-CGC GGA TCC TCT
CAG GTA CCC AGT T-3` (representing amino acids 23-29 and the
C-terminal sequences). PCRs (30 cycles) were carried out at 94 °C
for 1 min, 42 °C for 1 min, and 72 °C for 2 min. The PCR
products were then gel isolated and restricted with XmaI,
which cuts once between amino acids 23 and 29. The products were then
ligated and directly used for a second PCR reaction with primers
representing N-terminal and C-terminal sequences. p34 lacking amino
acids 2-30 (p342-30) was prepared by PCR using a set
of primers (5`-AGG ATC CAT GGC ACC TTC TCA AGC CGA A-3` and 5`-CGC GGA
TCC TCT CAG GTA CCC AGT T-3`) to generate the mutant under the same
conditions as described above. The PCR products, after gel isolation,
were cloned into the BamHI site of pVL941. p34 lacking 33
amino acids at the C terminus (p34
33C) was prepared similarly to
p34
2-30 except that the following primers were used: 5`-CGC
GGA TCC ATG TGG AAC-3` and 5`-AGG ATC CTT ACA TGT GTT TCA GCT GGT T-3`.
Baculovirus Infection, Metabolic Labeling, and
Immunoprecipitation
Insect (Sf9) cell culture and preparation of
recombinant baculoviruses have been described previously (Stigger
et al., 1994). Sf9 cells (2.0 10
) were
plated on a 60-mm dish and infected with each recombinant baculovirus
at a multiplicity of infection of 30 for 40 h at 27 °C. The cells
were then labeled for 4 h with Tran
S-labeled methionine at
200 µCi/ml (1000 Ci/mmol) in 1.5 ml of methionine-free medium/5%
dialyzed fetal calf serum and washed with phosphate-buffered saline
(PBS), prior to being lysed for 1 h on ice in 0.5 ml of EBC buffer (50
mM Tris-HCl at pH 8.0, 120 mM NaCl, 0.5% Nonidet
P-40, 1 mM DTT, 1 mM EDTA, 0.1 mM NaF, 10
mM
-glycerophosphate, 0.1 mM sodium
orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml
leupeptin, and 0.2 mg/ml antipain). Cleared cell lysates (50 µl)
were subsequently incubated overnight with 5 µg of purified
monoclonal antibody in the presence of bovine serum albumin (200
µg/ml) at 4 °C with rocking. Protein G-Sepharose was then
added, and the lysates were incubated for 1 h at 4 °C. Finally, the
immunoprecipitates were collected by centrifugation, washed five times
with EBC buffer, and then analyzed by SDS-PAGE.
Protein Isolation
Wild-type RPA and RPA mutants
were isolated from insect cells coinfected with recombinant
baculoviruses encoding p70, p11, and either wild-type or mutant p34
(p34(S/A:23,29), p3433C, or p34
2-30) as described
earlier (Stigger et al., 1994).
SV40 DNA Replication in Vitro
The reactions were
carried out as described by Wobbe et al.(1985). In brief, the
reaction mixtures (40 µl) included 40 mM creatine
phosphate-di-Tris salt (pH 7.7), 1 µg of creatine kinase, 7
mM MgCl, 0.5 mM DTT, 4 mM ATP,
200 µM UTP, GTP, and CTP, 100 µM dATP, dGTP,
and dCTP, 25 µM [
H]dTTP (300
cpm/pmol), 0.6 µg of SV40 T Ag, 0.23 µg of pSV01
EP, and
the indicated amounts of replication proteins or extracts. The
reactions ran for 1 h at 37 °C, after which the acid-insoluble
radioactivity was measured.
-
P]dCTP (30,000 cpm/pmol) instead of
[
H]dTTP in the reactions just described. After
incubation, the reactions were stopped by the addition of 80 µl of
a solution containing 20 mM EDTA, 1% sodium dodecyl sulfate,
and E. coli tRNA (0.5 mg/ml). DNA was isolated and
electrophoretically separated in a 1.2% alkaline-agarose gel (40
mM NaOH and 1 mM EDTA) for 12-14 h at 2 V/cm.
The gel was subsequently dried and exposed to x-ray film.
In Vitro Phosphorylation of RPA
Reaction mixtures
(30 µl) contained 40 mM creatine phosphate-di-Tris salt
(pH 7.7), 1 µg creatine kinase, 7 mM MgCl, 0.5
mM DTT, 5 µg of bovine serum albumin, 4 mM ATP,
200 µM UTP, GTP, and CTP, 100 µM dATP, dGTP,
dTTP, and dCTP, 1 mM Na
VO
, 10
mM NaF, and 0.6 µg of SV40 T Ag. Where indicated, 100
µg of ammonium sulfate fraction 35-65% (AS 35-65) of
HeLa cell cytosolic extract, 1.0 µg of RPA, or 0.3 µg of SV40
origin-containing DNA was included in the reactions which were
incubated at 37 °C for 1 h. Proteins were then separated by 11%
SDS-PAGE, transferred to nitrocellulose, immunoblotted with an anti-p34
polyclonal antibody (rabbit), and visualized by
I-protein
A autoradiography (Stigger et al., 1994).
DNA Polymerase
DNA pol Assay and SV40 DNA Unwinding
Assays
activity and the unwinding of SV40
origin-containing DNA (pSV01
EP) were assayed as described
previously (Lee et al., 1989; Stigger et al., 1994).
Interaction between RPA and SV40 T Ag
Protein
interaction was determined by the modified ELISA described by
Dornreiter et al.(1992). The 96-well plates were coated with
1.0 µg of either wild-type or mutant RPA overnight at 4 °C.
After washing with PBS, wells were blocked with 3% bovine serum albumin
in PBS for 1 h at 37 °C. The indicated amounts of SV40 T Ag were
added, and the wells were incubated for another hour at room
temperature before being washed extensively with PBS. RPA-bound SV40 T
Ag was measured by incubating the wells with a horseradish
peroxidase-conjugated SV40 T Ag monoclonal antibody (Pab 419) for 1 h
at 37 °C. Conjugation of the antibody to horseradish peroxidase
(Zymed Actizyme-Peroxidase kit) was done according to the
manufacturer's specifications. After extensive washing with PBS,
the chromogenic substrate ABTS acid and hydrogen peroxide were added,
and the colorimetric reaction was monitored at 410 nm.
ssDNA Binding Assay
The assay was performed
according to method used by Kim et al.(1992) with the
following modifications. The reaction mixture (20 µl) contained 50
mM Hepes-KOH (pH 7.5), 150 mM NaCl, 1 mM
MgCl, 0.5 mM DTT, 10% glycerol, 50 fmol of
5`-
P-labeled oligo(dT)
(2200 cpm/fmol), plus
the indicated amount of RPA, and was incubated for 15 min at 25 °C.
The complex was electrophoretically separated on a 5% polyacrylamide
gel in 0.5
TBE (89 mM Tris borate, 2 mM EDTA)
at 15 V/cm. The gel was then dried and exposed to x-ray film. To
quantitate the data, the protein-DNA complex bands were excised and
analyzed by liquid scintillation counting.
RPA p34 Mutants
To study the function of RPA p34
in DNA replication, we created two p34 deletion mutants and a p34
mutant that lacks the putative Cdk recognition sites (by serine to
alanine substitutions at amino acids 23 and 29). We first examined
whether mutant p34 can form a complex with other RPA subunits. After we
had coinfected Sf9 cells with recombinant baculoviruses encoding p34
(wild-type or mutant), p11, and p70, RPA complexes were
immunoprecipitated from cell lysates using an anti-p34 monoclonal
antibody or an anti-p70 antibody (Fig. 1). Both the N-terminal
and the C-terminal deletion mutants (p342-30 and
p34
33C, respectively) were able to form heterotrimeric complexes
with p70 and p11. Chromatographic behaviors and yields of these
deletion mutants were similar to those of wild-type RPA (data not
shown). p34
33C was immunoprecipitated by the anti-p70 antibody but
not by the anti-p34 monoclonal antibody, suggesting that the C terminus
of p34 (amino acids 238-270) contains the p34 monoclonal antibody
recognition site.
Figure 1:
Formation of wild-type and mutant RPA
complexes in vivo.
[S]Methionine-labeled lysates from insect (Sf9)
cells coinfected with recombinant baculoviruses encoding p70, p11, and
either wild-type or mutant p34 are indicated at the top of the figure.
RPA complexes were immunoprecipitated with an anti-p34 antibody
(lanes 1-4) or an anti-p70 antibody (lanes
5-8) as described under ``Experimental
Procedures.''
The N-terminal Region of RPA p34 Is Required for Its
Phosphorylation by Cdk and DNA-dependent Kinase
At least two
different kinases (cyclin-dependent kinase and DNA-dependent kinase)
can phosphorylate RPA p34 in vitro (Elledge, 1992; Dutta and
Stillman, 1992; Fotedar and Roberts, 1992; Pan and Hurwitz, 1993).
Therefore, we examined the phosphorylation of both wild-type and mutant
RPA p34 under replication conditions. The 35-65% ammonium sulfate
fraction (AS 35-65) of HeLa cell cytosolic extracts was used in
this assay because it contains all of the required host replication
factors except RPA (Wobbe et al., 1987). The amount of RPA
present in AS 35-65 was negligible compared to the exogenous RPA
(1.0 µg) in this experiment (Fig. 2, lane1). In the absence of SV40 origin-containing DNA, less
than 50% of wild-type RPA was phosphorylated and appeared as a slow
migrating band on the denaturing gel (Fig. 2, lane3). This slow migrating band, which disappears on
phosphatase treatment (data not shown), probably represents RPA that
was phosphorylated by cyclin A-Cdk2 (or another cyclin-dependent
kinase), because, under the same conditions, p34 mutants
(p34(S/A:23,29) and p34
2-30) lacking the major Cdk consensus
sites were not phosphorylated. p34(S/A:23,29) can be phosphorylated by
purified Cdk2-cyclin A in vitro, indicating that additional
Cdk target sites exist.(
)
In the presence of SV40
DNA, both wild-type RPA and RPA containing p34(S/A:23,29) were
efficiently phosphorylated by DNA-dependent kinase (indicated by the
highly retarded bands in lanes4 and 7 of
Fig. 2
). This suggests that the cyclin-dependent kinase
phosphorylation sites are different from those targeted by
DNA-dependent kinase. C-terminally deleted RPA p34 (p34
33C)
displayed a phosphorylation pattern similar to that of wild-type RPA
with the exception that this mutant was phosphorylated less efficiently
in the presence than in the absence of SV40 DNA (Fig. 2,
lanes 11-13). These data indicate that the C-terminal
region of p34 is not required for RPA p34 phosphorylation but may be
necessary for efficient replication. However, the p34
2-30
mutant was not phosphorylated under either conditions, as measured by
both immunoblotting (Fig. 2, lanes 8-10) and
assaying for kinase activity (data not shown), indicating that the
N-terminal region of p34 is necessary for its phosphorylation by both
kinases. This region contains 5 serine residues (amino acids 4, 8, 11,
12, and 13), in addition to the major consensus sites for Cdk (amino
acids 23 and 29).
Figure 2:
In vitro phosphorylation of wild-type and
mutant RPA p34. Reaction conditions were described in the experimental
procedures. Where indicated, 1.0 µg of either wild-type or mutant
RPA, 100 µg of an ammonium sulfate fraction (35-65%) of HeLa
cell cytosolic extract, or 0.3 µg of SV40 origin-containing DNA
were included. After the reactions, RPA p34 was visualized using an
anti-p34 polyclonal antibody (rabbit).
The C-terminal but Not the N-terminal Region of RPA p34
Is Required for Efficient SV40 DNA Replication
In vivo,
RPA p34 is phosphorylated at the G/S boundary and remains
phosphorylated during S-phase, suggesting that p34 phosphorylation may
be important to DNA replication. Since p34
2-30 is not
phosphorylated by kinases present in HeLa cell cytosolic extracts, this
mutant should help determine whether RPA p34 phosphorylation is
directly involved in the replication process.
2-30 or p34(S/A:23,29)
functioned as efficient as wild-type RPA, when added to the AS
35-65 fraction, in our SV40 DNA replication system (Fig. 3,
panelA), whereas p34
33C poorly supported DNA
replication. We also examined the function of these mutants in the SV40
monopolymerase system which contains pol
-primase, topo I, and
SV40 T Ag (Fig. 3, panelB). Similarly to SV40
replication with crude extracts (AS 35-65), p34
33C supported
only about 20% of the activity supported by wild-type RPA or the other
two mutants. This result indicates that RPA p34 requires its C-terminal
domain, but not phosphorylation, to function effectively in an SV40 DNA
replication system.
Figure 3:
Comparison between wild-type and mutant
RPA function in SV40 DNA replication in vitro.A,
SV40 DNA replication in vitro with crude extracts. Replication
reactions comprised SV40 origin-containing DNA (pSV01EP), SV40 T
Ag, the 35-65% ammonium sulfate fraction (AS 35-65) of HeLa
cell cytosolic extract (100 µg), [
H]dTTP, and
the indicated amounts of either wild-type or mutant RPA. Reaction
mixtures were incubated for 1 h at 37 °C, and the reaction products
examined for acid-insoluble radioactivity. B, SV40 DNA
replication in vitro with purified proteins (monopolymerase
system). The replication reaction mixture contained 0.4 µg of SV40
T Ag, pol
-primase complex (0.2 and 0.05 units, respectively),
topo I (500 units), and the indicated amounts of wild-type or mutant
RPA, as well as [
-
P]dCTP instead of
[
H]dTTP. Reactions were incubated for 90 min at
37 °C and examined for acid-insoluble
radioactivity.
RPA p34 Is Involved in the Initiation Stage but Not in
the Elongation Stage of DNA Replication
We then examined which
stages of DNA replication are affected by p3433C by comparing the
kinetics of SV40 DNA replication in systems containing AS 35-65
fractions supplemented with wild-type RPA or RPA containing
p34
33C. The replication components were mixed, incubated at 37
°C, and aliquots were withdrawn at the times indicated in
Fig. 4
. PanelA shows the product sizes after
alkaline-agarose gel electrophoresis, and panelB indicates the rate of trichloroacetic acid-precipitable DNA
synthesis. DNA synthesis was slower in the reactions containing
p34
33C than in those containing wild-type p34. However, the
product sizes in both sets of reactions were comparable, indicating
that the mutant affected DNA synthesis at the level of initiation
rather than elongation.
Figure 4:
Rate of SV40 DNA synthesis in the presence
of wild-type or mutant RPA. Replication mixtures, containing
pSV01EP, SV40 T Ag, AS 35-65, and 0.6 µg of either
wild-type or mutant RPA, were prepared on ice, and incubated at 37
°C at time 0. Reactions in lanes7 and 14 lacked SV40 T Ag, and the reaction in lane15 contained no RPA. At the indicated times, aliquots were withdrawn
and product sizes were assessed by 1.2% alkaline-agarose gel
electrophoresis (panelA). Nucleotide incorporation
was measured by acid-insoluble radioactivity (panel B).
ssl and ssc represent the single-stranded linear and
single-stranded circular positions of pSV01
EP,
respectively.
To examine this further, replication
reactions including either wild-type RPA or RPA containing p3433C
were preincubated at 37 °C for 20 min. dNTPs were then added to
measure the rate of chain elongation following initiation
(Fig. 5). Despite the lower level of total DNA synthesis in
mutant RPA-containing reactions compared to that in wild-type
RPA-containing reactions, the rate of elongation was again comparable
under both conditions.
Figure 5:
Comparison between wild-type and mutant
RPA function in chain elongation during SV40 DNA replication in
vitro. Reaction mixtures were preincubated in the absence of dNTPs
at 37 °C for 20 min. [-
P]dNTPs were
then added (time 0), and aliquots were removed at the indicated times.
Samples were analyzed by 1.2% alkaline-agarose gel electrophoresis for
DNA chain elongation. All other conditions were the same as those
described in the legend for Fig. 4; where indicated, 0.6 µg of
either wild-type or mutant RPA was added. Reactions in lanes6 and 12 were identical to those in lanes5 and 11, respectively, with the exception that
SV40 T Ag was omitted from the reactions in lanes6 and 12. ssl and ssc represent the
single-stranded linear and single-stranded circular positions of
pSV01
EP, respectively; n.t. denotes product lengths in
nucleotides.
Functional Analysis of RPA Containing
p34
To determine a specific role for RPA p34 in DNA
replication, we compared the behavior of RPA containing p3433C
33C
with that of wild-type RPA in several RPA-dependent assays: the
stimulation of pol
activity, the unwinding of SV40
origin-containing DNA, SV40 T Ag interaction, and binding to ssDNA.
to stimulate the elongation of primed template DNA(Erdile
et al., 1990). However, in the presence of the other two
subunits, p34 and p11, the functional activity of p70 may be altered.
Therefore, we have examined the effect of wild-type RPA as well as RPA
containing p34
33C on pol
activity using
poly(dA)
oligo(dT)
(Fig. 6). Under
these conditions, both wild-type and mutant RPA stimulated pol
activity by about 5-6-fold, indicating that the poor replication
activity of RPA containing p34
33C is not related to its ability to
stimulate pol
.
Figure 6:
Effect of wild-type and mutant RPA on DNA
pol activity. The indicated amount of either wild-type or mutant
RPA (RPA:p34
33C and RPA:p34
2-30) was added to the
reaction mixtures, which included 0.1 unit of human pol
, 4
mM ATP, and 0.1 µg of
poly(dA)
oligo(dT)
. Incubations ran for 60
min at 37 °C.
With the exception of T4 gene 32, RPA from
other sources (yRPA, E. coli SSB, and adenovirus DBP) can
replace human RPA in SV40 unwinding assays (Kenny et al.,
1989), suggesting that the role of RPA in DNA unwinding is not merely
to stabilize single-stranded DNA. When we compared the activities of
wild-type and mutant RPAs in our unwinding assay, we found that as we
increased the amount of RPA, the reaction that included
p342-30 showed an unwinding pattern similar to that obtained
with wild-type RPA. However, much less DNA unwinding was observed in
the reaction containing p34
33C (Fig. 7). This latter result
could explain why SV40 DNA replication with the p34
33C mutant is
so inefficient.
Figure 7:
Comparison between the ability of
wild-type and mutant RPA to unwind SV40 origin-containing DNA. The
unwinding assay was carried out in the presence of 0.6 µg of SV40 T
Ag, topoisomerase I (1500 units), and 0.3 µg (lanes2, 6, and 10), 0.6 µg (lanes3, 7, and 11), and 1.2 µg
(lanes4, 5, 8, 9,
12, and 13) of either wild-type or mutant RPA as
indicated on the top of the figure. U indicates the position
of unwound DNA. I` and II represent the positions of
relaxed replicative form I DNA (RFI`) and nicked DNA (RFII),
respectively.
To investigate this further, we examined RPA's
ability to bind to ssDNA and to interact with SV40 T Ag: two RPA
functions that are likely to be involved in DNA unwinding. A fixed
amount of wild-type or mutant RPA was bound to an ELISA plate and then
incubated with various amounts of SV40 T Ag. As reported previously
(Dornreiter et al., 1992), wild-type RPA interacted with SV40
T Ag in this ELISA assay (Fig. 8). RPA containing
p342-30 (which lacks p34 phosphorylation sites) interacted
with SV40 T Ag to a similar degree as wild-type RPA. RPA containing
p34
33C, however, did not interact properly with SV40 T Ag. The low
level of signal obtained in this assay with T Ag and RPA containing
p34
33C consistently leveled off at low concentrations of mutant
RPA, indicating that this low level of signal is probably not due to
protein interactions.
Figure 8:
Interaction of wild-type and mutant RPA
with SV40 T Ag. Wild-type or mutant RPA-coated ELISA plates were
incubated with various amounts of SV40 T Ag for 1 h at 37 °C. SV40
T Ag bound to RPA was detected with a peroxidase-conjugated SV40 T Ag
monoclonal antibody (Pab 419) as described under ``Experimental
Procedures.''
When we compared wild-type RPA and mutant RPA
(RPA containing p3433C) for ssDNA binding activity using a gel
shift assay with oligo(dT) as a substrate (Kim et al., 1992;
Blackwell and Borowiec, 1994), we found, as others have (Blackwell and
Borowiec, 1994), that two distinct bands were obtained. At low
concentrations (less than 50 ng), wild-type RPA bound ssDNA
2-3-fold better than the mutant, but at a high RPA concentration
(1000 ng) both wild-type and mutant RPA showed similar binding
affinities (Fig. 9). Furthermore, at the higher concentration,
most of the wild-type RPA-DNA complex appeared as a slow migrating band
(form II), whereas the mutant RPA-DNA complexes appeared as forms I and
II. We have observed similar results using oligo(dT)
as
the substrate in place of oligo(dT)
(data not shown).
Figure 9:
ssDNA binding activities of wild-type and
mutant RPA. Various amounts of either wild-type or mutant RPA were
incubated with 50 fmol of 5`-P-labeled oligo(dT)
for 15 min at 25 °C. The protein-DNA complexes were then
separated from unbound DNA by 5% polyacrylamide
(acrylamide:bisacrylamide = 29:1) gel
electrophoresis.
2-30, which lacks the
phosphorylation sites targeted by Cdk2-cyclin A and DNA-dependent
kinase; and p34
33C, which lacks the region recognized by our
anti-p34 monoclonal antibody that neutralizes SV40 DNA replication
in vitro. Although deletion of the N-terminal amino acids of
p34 completely abolished its phosphorylation by both Cdk and
DNA-dependent kinase, a double point mutant of p34 (p34(S/A:23,29)),
which lacks the major Cdk phosphorylation sites, was efficiently
phosphorylated by DNA-dependent kinase. This suggests that the
phosphorylation of RPAp34 by DNA-dependent kinase is independent of its
phosphorylation by Cdk. The 5 serine residues (amino acids 4, 8, 11,
12, and 13) in the N-terminal region of p34 may be the targets for
DNA-dependent kinase since the mutant lacking this region
(p34
2-30) could not be phosphorylated by either kinase.
Alternatively, the physical presence of the N-terminal region may be
necessary for the kinase to phosphorylate RPA p34, rather than to
directly supply phosphorylation sites. Substitution mutants (serine to
alanine) will be needed to clarify this point.
extracts so that they can support SV40 DNA replication
(D'Urso et al., 1990; Dutta and Stillman, 1992). Our
data strongly suggest that RPA p34 phosphorylation, by either Cdk or
DNA-dependent kinase, is not required for SV40 DNA replication in
vitro. It should be pointed out, however, that SV40 is a minimal
model and that RPA p34 phosphorylation may be involved in more
complicated systems such as chromosomal DNA replication.
33C was 2-3-fold lower than that of wild-type RPA
especially at low concentrations of RPA. More experiments will be
necessary to clarify the role of RPA p34 in DNA binding.
activity.
However, this antibody has no significant effect on the unwinding of
SV40 origin-containing DNA (Kenny et al., 1990). In our
experiments, mutant RPA that lacks the anti-p34 antibody recognition
site (the C-terminal region of RPA p34), supports SV40 DNA replication
poorly, presumably because it fails to interact with SV40 T Ag. The
same mutant, however, can stimulate pol
activity. These results
could be explained as steric hindrance due to the antibody binding to
p34 and blocking the p70 domain, which is necessary for pol
stimulation. If the antibody cannot bind, the p70 domain is exposed and
can interact with pol
. Similarly, although the p34 antibody had
no effect on SV40 DNA unwinding, mutant RPA containing C-terminally
deleted p34 did inhibit DNA unwinding. This discrepancy can be
explained by the fact that the deleted region contains both the
antibody recognition site and the region necessary for efficient SV40
DNA unwinding. These two domains are, however, located at different
sites within the deleted region.
and
, DNA polymerase
and
,
respectively; topo, topoisomerase; PCR, polymerase chain reaction; RPA,
replication protein A; SSB, single stranded DNA-binding protein; ssDNA,
single-stranded DNA; T Ag, SV40 large tumor antigen; DTT,
dithiothreitol; TBE, Tris borate-EDTA buffer; PBS, phosphate-buffered
saline; ELISA, enzyme-linked immunosorbent assay; Cdk, cyclin-dependent
kinase; kb, kilobase pair(s); ABTS,
2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.