©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro(*)

Suk-Hee Lee (1)(§), Dong Kyoo Kim (¶)

From the (1) Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318 and the Department of Pathology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 , 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.


INTRODUCTION

The in vitro simian virus 40 (SV40)() 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).

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 -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).

RPA p34 is phosphorylated on serine residues at the G/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.

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/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.

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.


EXPERIMENTAL PROCEDURES

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 (p3433C) was prepared similarly to p342-30 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 TranS-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 p342-30) 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 pSV01EP, 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.

Replication products were analyzed using [-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 NaVO, 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 Assay and SV40 DNA Unwinding Assays

DNA pol activity and the unwinding of SV40 origin-containing DNA (pSV01EP) 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.


RESULTS

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 p3433C, 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). p3433C 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 p342-30) 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 (p3433C) 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 p342-30 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 p342-30 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.

We, therefore, examined whether our RPA p34 mutants supported SV40 DNA replication in vitro. RPA containing p342-30 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 p3433C 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), p3433C 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 p3433C. 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 p3433C 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 pSV01EP, 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 pSV01EP, respectively; n.t. denotes product lengths in nucleotides.



Functional Analysis of RPA Containing p3433C

To determine a specific role for RPA p34 in DNA replication, we compared the behavior of RPA containing p3433C 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.

Previously, the p70 subunit of RPA alone was shown to be required by pol 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 p3433C 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 p3433C 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:p3433C and RPA:p342-30) 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 p3433C (Fig. 7). This latter result could explain why SV40 DNA replication with the p3433C 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 p3433C, however, did not interact properly with SV40 T Ag. The low level of signal obtained in this assay with T Ag and RPA containing p3433C 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.




DISCUSSION

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: p342-30, which lacks the phosphorylation sites targeted by Cdk2-cyclin A and DNA-dependent kinase; and p3433C, 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 (p342-30) 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.

It has been suggested that RPA phosphorylation by Cdk is involved in the activation of G 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.

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 p3433C 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.

The anti-p34 monoclonal antibody inhibits both in vitro SV40 DNA replication and the stimulatory effect of RPA on pol 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.


FOOTNOTES

*
This research was supported by Grant IN-176-C from American Cancer Society, Cancer Center Support Grant 5P30 CA 21765-16, and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38101-0318. Tel.: 901-531-2519; Fax: 901-523-2622; E-mail: lee@mbcf.stjude.org.

Supported by a fellowship from Inje Foundation, Kimhae, Korea.

The abbreviations used are: SV40, simian virus 40; pol 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.

S.-H. Lee, unpublished data.


ACKNOWLEDGEMENTS

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.


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