Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

Mammalian replication protein A (RPA) undergoes DNA damage-dependentphosphorylation at numerous sites on the N terminus of the RPA2subunit. To understand the functional significance of RPA phosphorylation,we expressed RPA2 variants in which the phosphorylation siteswere converted to aspartate (RPA2_D) or alanine (RPA2_A). AlthoughRPA2_D was incorporated into RPA heterotrimers and supportedsimian virus 40 DNA replication in vitro, the RPA2_D mutant wasselectively unable to associate with replication centers invivo. In cells containing greatly reduced levels of endogenousRPA2, RPA2_D again did not localize to replication sites, indicatingthat the defect in supporting chromosomal DNA replication isnot due to competition with the wild-type protein. Use of phosphospecificantibodies demonstrated that endogenous hyperphosphorylatedRPA behaves similarly to RPA2_D. In contrast, under DNA damageor replication stress conditions, RPA2_D, like RPA2_A and wild-typeRPA2, was competent to associate with DNA damage foci as determinedby colocalization with ${gamma}$ -H2AX. We conclude that RPA2 phosphorylationprevents RPA association with replication centers in vivo andpotentially serves as a marker for sites of DNA damage.

INTRODUCTION

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Introduction

DNA-damaging stress leads to the inception of a variety of cellularresponses that serve to minimize mutation and prevent genomicinstability. In particular, the cell cycle checkpoint apparatusis activated to block S phase entry and, in those cells in thereplicative phase, to both inhibit firing of late origins ofDNA replication and avert the collapse of replication forksblocked by damage (3). The DNA repair machinery is mobilizedin concert to repair lesions and to allow eventual restart ofstalled replication forks. One factor that plays essential rolesboth during DNA replication and in the repair- and recombination-mediatedrecovery from damage is replication protein A (RPA), the eukaryoticsingle-stranded (ss) DNA-binding protein (27, 52).

RPA is a heterotrimeric protein consisting, in mammalian cells,of $~$ 70- (RPA1), 30- (RPA2), and 14 (RPA3)-kDa subunits. DuringDNA replication, RPA acts at the fork, stabilizing ssDNA andfacilitating nascent strand synthesis by the replicative DNApolymerases. Under DNA-damaging conditions, RPA-ssDNA complexesact to recruit and activate a key checkpoint mediator consistingof the ATR and ATRIP (ATR-interacting protein) protein-kinasecomplex (54). At DNA damage-dependent nuclear foci, RPA interactswith repair and recombination components to process double-strandDNA breaks and other lesions (19). RPA activity is regulatedby various stress conditions. In particular, heat shock (12,47, 48), exposure to UV radiation (9), and treatment with DNA-alkylatingagents (30) each cause the generation of an RPA species thatis unable to support DNA replication in vitro. In the case ofheat shock, the inhibition of RPA activity is mediated by astress-dependent association with the nucleolar protein nucleolin(12, 47).

In an area with potential regulatory significance, RPA undergoesboth stress-dependent and -independent phosphorylation on theextreme N terminus of the RPA2 subunit. A basal level of RPAmodification by cyclin-cdk complexes occurs at two sites (16,35). Following stress, such as exposure to ionizing (31) orUV (9) radiation, or treatment with radiomimetic agents, suchas camptothecin (CPT) (42), human RPA2 can be phosphorylatedat five or more additional sites out of a possible seven bythe phosphatidylinositol 3-kinase-related kinases (PIKKs) DNA-PK,ATM, and perhaps ATR (7, 10, 17, 18, 31, 33, 35, 46, 53). ATMand ATR are activated in response to DNA damage and replicationstress, and they modify various effectors that facilitate thedamage and cell cycle checkpoint responses (1). DNA-PK is requireddirectly in the repair of double-strand DNA breaks and in V(D)Jrecombination (15). These data could suggest that the functionof stress-dependent modification of RPA is to repress DNA replicationor to promote recovery from DNA damage, but there are as yetno compelling data for either role. While the results of certainstudies suggest that RPA modification by PIKKs may lead to theinhibition of DNA replication in vitro and in vivo (9, 37),direct testing of this possibility has not shown any appreciableeffects of RPA phosphorylation on binding to ssDNA or on replicationin vitro using a simian virus 40 (SV40)-based assay (7, 23).

Because previous work has primarily studied the effects of mammalianRPA phosphorylation using in vitro systems, it is possible thatthe modulation of RPA activity by phosphorylation might be observedonly in the cellular milieu. Testing this hypothesis, we foundthat RPA2 phosphorylation mutants that mimic the hyperphosphorylatedform were unable to localize to replication centers in normalcells. Interestingly, binding of the hyperphosphorylation mimicto DNA damage foci was unaffected, as determined by colocalizationwith the DNA damage marker ${gamma}$ -H2AX. Similar behavior was observedwith endogenous hyperphosphorylated RPA. We conclude that RPAphosphorylation following damage both prevents RPA from catalyzingDNA replication and potentially serves as a marker to recruitrepair factors to sites of DNA damage.

MATERIALS AND METHODS

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Materials and Methods

Cell lines and stress treatments. U2-OS and HeLa cells were maintained in McCoy's 5 M and Dulbecco'smodified Eagle's media, respectively, supplemented with 10%fetal bovine serum and 50 µg of gentamicin/ml. When theeffect of stress was examined, the cells were treated with either1 µM CPT (Sigma) for 1 or 3 h, 2.5 mM hydroxyurea (HU;Sigma) for either 1 or 3 h, or 7 µM aphidicolin (Sigma)for 3 h. To inhibit the cellular checkpoint response, cellswere treated with 5 mM caffeine for 30 min prior to stress.Transfection experiments were performed using Effectene (Qiagen).

Generation of RPA2 mutant constructs. To generate the myc-RPA2_wt and myc-RPA2_D mammalian expressionvectors, the human RPA2 genes from plasmids p11dtRPA and p11dtRPA· 32Asp8 (4, 24) were inserted into the XbaI and BstBIsites of the pEF6/Myc-HisA vector (Invitrogen), resulting inpERPA2wt and pERPA2D. Expression of the His₆ tag from pEF6/Myc-HisAwas prevented by mutating the ATG codon at position 1863 toa TGA codon. Vectors expressing the intermediate RPA2 phosphorylationmutants and RPA2_A were constructed by a combination of site-directedmutagenesis of either pERPA2wt or pERPA2D (as appropriate) atpositions 23, 29, and 33 and replacement of larger segmentsof the RPA2 N terminus with synthetic oligonucleotides encodingmutant phosphorylation regions. Detailed cloning proceduresare available upon request.

Protein purification and in vitro replication assay. The RPA_RPA2wt and RPA_RPA2D heterotrimers were expressed in Escherichiacoli BL21 transformed with p11dtRPA and p11dtRPA · 32Asp8,respectively, and purified as described previously (24, 26).The SV40 large tumor (T) antigen used for SV40 DNA replicationreactions was prepared from extracts of Sf9 cells infected withthe recombinant baculovirus Ac941SVT (5) and purified usingimmunoaffinity chromatography (6). The AS65 fraction lackingRPA was prepared from HeLa cell extracts by ammonium sulfatefractionation according to the method of Wobbe et al. (51).SV40 DNA replication reaction mixtures (50 µl) containing40 mM creatine phosphate (diTris salt; pH 7.8); 7 mM MgCl₂;4 mM ATP; 25 µg of creatine kinase/ml; 0.4 mM dithiothreitol;200 µM (each) CTP, GTP, and UTP; 100 µM (each) dATP,dGTP, and dCTP; 25 µM [³H]dTTP ( $~$ 500 cpm/pmol); 0.2 µgof the ori-containing plasmid pSV01 ${Delta}$ EP (50); 200 µg ofthe AS65 fraction; 0 to 700 ng of RPA_RPA2wt or RPA_RPA2D; and750 ng of SV40 T antigen were incubated at 37°C for 2 h.Replication activity was determined by precipitating the high-molecular-weightDNA with trichloroacetic acid and quantitating the amount of³H in the precipitate by scintillation counting. To examinethe DpnI resistance of the replication products, replicationreaction mixtures containing 600 ng of either RPA_RPA2wt or RPA_RPA2Dand 100 µM [ ${alpha}$ -³²P]dCTP (1,000 cpm/pmol) to label the replicationproducts were incubated at 37°C for 2.5 h. Following removalof protein by phenol extraction, the DNA products were firstlinearized by digestion with PstI and then either mock treatedor incubated with 2.5 U of DpnI to cleave nonreplicated DNA.The digestion products were separated by electrophoresis througha 1.1% agarose gel and visualized both by ethidium bromide stainingand by autoradiography.

Immunoprecipitation and immunoblotting. Transfected U2-OS cells were lysed in lysis buffer (50 mM Tris-HCl,pH 7.4, 150 mM NaCl, 1% [vol/vol] NP-40, 1 mM phenylmethylsulfonylfluoride, 0.1 mM Na₃VO₄, 1 mM NaF, and 1 µg each of aprotinin,leupeptin, and pepstatin per ml). The cell extracts were thenincubated at 4°C for 2 h with 70A anti-RPA1 monoclonal antibody(28) conjugated to CNBr-activated Sepharose beads (AmershamBiosciences). The immunoprecipitate was washed five times withlysis buffer and resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) (13% [wt/vol] polyacrylamide).To test RPA2 phosphorylation and myc-RPA2 expression, cellswere directly lysed in SDS-PAGE sample buffer and proteins wereseparated by SDS-PAGE. For phosphatase treatment, cells werelysed in ${lambda}$ protein phosphatase buffer (New England Biolabs) containing1% Triton X-100 and 1 µg each of aprotinin, leupeptin,and pepstatin per ml. Cell lysates ( $~$ 20 µg of protein)were then incubated with 400 U of ${lambda}$ protein phosphatase for 30min at 30°C or mock treated in the presence of protein phosphataseinhibitors (0.1 mM Na₃VO₄, 1 mM NaF). The Western blots weredeveloped with an anti-RPA2 34A mouse monoclonal antibody (28)or a rabbit polyclonal anti-pSer4/pSer8-RPA2 antibody obtainedfrom Bethyl Laboratories, Inc. (Montgomery, Tex.). Proteinswere detected using enhanced chemiluminescence (Amersham Biosciences).

Cell cycle assay. Forty-eight hours posttransfection, cells were incubated with10 µM bromodeoxyuridine (BrdU). After a 30-min incubation,the cells were fixed and processed according to the BrdU FlowKit manual (BD Pharmingen). Following incubation with rat anti-BrdU(Harlan Sera-Lab) and rabbit anti-myc (Upstate Biotechnology)antibodies, the cells were stained with anti-rat fluoresceinisothiocyanate-conjugated and anti-rabbit phycoerythrin-conjugatedsecondary antibodies (Jackson ImmunoResearch Laboratories).The DNA was stained with 7-aminoactinomycin D, and the cellswere subjected to fluorescence-activated cell sorting (FACS)analysis.

Immunofluorescence microscopy. Transfected cells were processed by two methods. To test proteinexpression and transfection efficiency, the cells were firstwashed with phosphate-buffered saline (PBS), fixed with 4% (wt/vol)formaldehyde in PBS for 15 min at room temperature, and thenextracted with PBS containing 0.5% (vol/vol) Triton X-100 for5 min. To study chromatin-bound proteins, the cells were extractedwith 0.5% (vol/vol) Triton X-100 for 5 min prior to fixationas described previously (13). When required, cells were incubatedin media containing 10 µM BrdU for 10 min prior to harvest.For detection of incorporated BrdU, DNA was denatured with HClusing standard procedures. RPA2 silencing was achieved usinga short interfering RNA (siRNA) duplex targeted to the 5'-CCUAGUUUCACAAUCUGUUsequence found in the 3' noncoding region of RPA2 mRNA. Preparedcells were incubated, as required, with rabbit anti-myc (UpstateBiotechnology), mouse anti-RPA1 70A and anti-RPA2 34A (28),rabbit anti-pSer4/pSer8-RPA2 (Bethyl Laboratories), rat anti-BrdU(Harlan Sera-Lab), and mouse anti- ${gamma}$ H2AX (Upstate Biotechnology)antibodies. Following staining with the appropriate secondaryantibodies (Jackson ImmunoResearch Laboratories), the cellswere examined by epifluorescence microscopy using a Zeiss Axiophotmicroscope. To calculate the relative frequency of myc-RPA2-positivecells (see Fig. 6H and 8M), the fraction of cells transfectedwith myc-RPA2_wt or the myc-tagged RPA mutants was first determinedby processing cells without prior Triton X-100 extraction (e.g.,F_{transfection:wt} and F_{transfection:D4}). Separately, the fractionof cells showing significant chromatin staining was also determined(e.g., F_chromatin:wt and F_chromatin:D4). The relative frequencyof cells that were positive, for example, for myc-RPA2_D4 chromatinstaining was calculated using the following formula: relativefrequency = (F_chromatin:D4/F_{transfection:D4})/(F_chromatin:wt/F_{transfection:wt})· 100%. Each value determined was the result of threeindependent experiments.

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FIG. 6. Increase in negative charge at the RPA2 N terminus decreases frequency of myc-positive cells. (A to F) Cells were transfected with myc-RPA2_wt or myc-RPA2_D41 as indicated. Representative epifluorescence images showing total myc-RPA2 staining (no extraction [A and B]), chromatin-bound myc-RPA2 (C and D), and chromatin-bound RPA1 (E and F) are shown. (G and H) Serines and threonine (boldface and underlined) at the potential phosphorylation sites in the N terminus of the RPA2 subunit were replaced with alanines (A) or aspartates (D) as indicated. Cells expressing these myc-tagged RPA2 mutants were then analyzed by immunofluorescence microscopy for the presence of myc-RPA2 bound to chromatin and, in parallel reactions, for transfection efficiency. The relative frequencies of myc-RPA2 positive cells were calculated as described in Materials and Methods.

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FIG. 8. Collapse of DNA replication forks stimulates RPA loading to damaged DNA. (A to H) U2-OS cells were transfected with plasmids expressing either myc-RPA2_wt or myc-RPA2_D as indicated for 48 h. The cells were then treated with 2.5 mM HU for either 1 (A and E) or 3 (B and F) h in the absence of caffeine or treated with HU for 1 (C and G) or 3 (D and H) h in the presence of 5 mM caffeine. The cells were Triton X-100 extracted and fixed and then stained for myc-RPA. (I to L) Colocalization of myc-RPA2_D with sites of DNA damage. U2-OS cells were transfected with a myc-RPA2_D-expressing plasmid. Forty-eight hours posttransfection, the cells were treated with 2.5 mM HU for 3 h and then extracted with 0.5% Triton X-100 and fixed. The cells were stained with ${gamma}$ -H2AX (I) and myc-RPA (J). The staining pattern of a representative cell and the image of the merged staining patterns (K) are provided. (L) One section (boxed) of the composite image is shown enlarged to reveal the degree of signal overlap. (M) Graph showing the fractions of myc-RPA2-transfected cells with a significant chromatin-bound myc-RPA2_wt (green bars) or myc-RPA2_D (brown bars) signal. The fractions of cells showing chromatin-bound RPA were quantified by visual inspection of 100 to 200 cells. The bar graph values were calculated as described in Materials and Methods. Note that although RPA2_wt staining is consistently detected in a greater fraction of cells than RPA2_D staining, this result is expected because RPA2_wt can be observed both at replication centers and at DNA damage foci while RPA2_D localizes only to damage foci. (N) Effects of stress and caffeine treatment on endogenous RPA2 phosphorylation. U2-OS cells were mock treated (mock) or treated with either 1 µM CPT for 1 or 3 h, 2.5 mM HU for 1 or 3 h, 5 mM caffeine (c) for 3 h, 2.5 mM HU for 1 or 3 h in the presence of 5 mM caffeine or with 7 µM aphidicolin (A) for 3 h and 7 µM aphidicolin for 1 or 3 h in the presence of 5 mM caffeine. The band labeled RPA2' represents nonphosphorylated RPA, while P-RPA2 indicates the position of phosphorylated RPA.

RESULTS

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Results

The RPA2_D phosphorylation mimic localizes to the nucleus but is not chromatin bound. To understand the functional significance of RPA phosphorylation,we generated various human RPA2 constructs in which subsetsof the nine potential N-terminal phosphorylation sites weremutated. Previous studies have shown that two of the RPA2 sites(S23 and S29) are phosphorylated in a cell cycle-dependent mannerby cyclin-cdk2 complexes (16, 35). At least five of the otherseven (S4, S8, S11, S12, S13, T21, and S33) can be phosphorylatedin response to UV irradiation (53). Ionizing irradiation andtreatment with the radiomimetic agent CPT cause similar RPAhyperphosphorylation and likely modification of most if notall of these same sites (31, 42). Various data strongly suggestthat the PIKK members DNA-PK and ATM, and likely ATR, can independentlymodify the RPA stress-dependent sites (7, 10, 17, 18, 31, 33,35, 46), although only two (T21 and S33) have canonical SQ/TQsequences that are PIKK targets (1). Both of the cyclin-cdk2sites and six of the stress-dependent sites (S8, S11, S12, S13,T21, and S33) were replaced by aspartate to mimic phosphate(generating the RPA2_D mutant; see Fig. 6G for a schematic showingthe construction of this and other mutants). Although an aspartateresidue is not the same as phosphoserine or phosphothreonine,the use of aspartate residues to imitate phosphate has beenshown in many cases to have identical effects on protein structureand activity (25, 49). In the RPA2_A mutant, these same eightsites were converted to alanines to prevent phosphorylation(see also Fig. 6G). All of the mutants and the wild-type RPA2control (RPA2_wt) contained a C-terminal myc tag.

The RPA2_wt subunit was expressed in human U2-OS cells. To detectthe chromatin-bound fraction of RPA2, transfected cells wereextracted with nonionic detergent prior to formaldehyde fixation(13). Under such conditions, RPA bound to chromatin in nuclearreplication foci can be selectively visualized. The transfectedRPA2_wt subunit nearly completely colocalized with the endogenousRPA1 and exhibited a punctate distribution throughout the nucleus,consistent with its recruitment to DNA replication centers (Fig.1A to D). To confirm this observation, transfected cells werepulse-labeled with BrdU prior to fixation, and the sites ofRPA2_wt localization and BrdU incorporation were examined. Asexpected, the RPA2_wt subunit showed nearly complete colocalizationwith replicating chromatin (Fig. 1E to H). Taken together, theseresults indicate that the recombinant RPA2_wt subunit can functionallyreplace endogenous RPA2 in supporting chromosomal DNA replication.

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FIG. 1. The myc-RPA2_wt subunit colocalizes with endogenous RPA1 and DNA replication centers. U2-OS cells were transfected with a vector expressing myc-RPA2_wt. To allow visualization of chromatin-bound myc-RPA, the cells were first extracted with 0.5% Triton X-100 for 5 min and then fixed with formaldehyde. (E to H) To detect sites of DNA replication, BrdU was added to the medium 10 min before the cells were prepared for epifluorescence microscopy. As indicated, the cells were then stained with anti-myc (A and F), anti-RPA1 (B), or anti-BrdU (E) antibody. The extent of myc-RPA2_wt and endogenous RPA1 colocalization is shown (C), with enlargement of a particular nuclear region (boxed) (D). BrdU and myc-RPA2_wt colocalization are similarly shown (G and H).

We next examined the localization of the RPA2_A and RPA2_D mutants.Transfected cells were examined both without and with priordetergent extraction to reveal transfection efficiency and toshow the fraction bound to chromatin, respectively. The distributionof RPA2_A on chromatin (Fig. 2L) was virtually identical to thereplication pattern seen with endogenous RPA2 (data not shown)and the RPA2_wt variant (Fig. 2D) and showed nearly completecolocalization with endogenous RPA1 (Fig. 2K and data not shown).RPA2 phosphorylation is therefore not required for associationwith replication centers.

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FIG. 2. Lack of association of the RPA2_D mutant with chromatin in unstressed cells. U2-OS cells were transfected with a vector expressing myc-RPA2_wt (A to D), myc-RPA2_D (E to H), or myc-RPA2_A (I to L). (C, D, G, H, K, and L) To allow visualization of chromatin-bound myc-RPA, cells were first extracted with 0.5% Triton X-100 for 5 min and then fixed with formaldehyde (+ extraction). (A, B, E, F, I, and J) To assay for transfection efficiency, cells were also fixed without prior extraction (- extraction). The cells were then stained with anti-myc (B, D, F, H, J, and L) or anti-RPA1 (A, C, E, G, I, and K) antibody.

In dramatic contrast, we did not observe chromatin stainingfor RPA2_D (Fig. 2H), even though the RPA2_D mutant was expressedto an equivalent similar to that of the RPA2_wt construct (Fig.2F and B, respectively). Similar experiments were performedwith RPA2_D and RPA2_wt expressed as fusion proteins with greenfluorescent protein. While a modest RPA2_wt signal was detected,we did not observe an appreciable level of chromatin bindingfor RPA2_D (data not shown). We therefore find that mutationof RPA2 to a hyperphosphorylation mimic greatly reduces itslocalization to DNA replication centers.

In addition to the possibility that phosphorylation of RPA inhibitsits normal participation at the DNA replication fork in vivo,other explanations exist. One is that the myc-RPA2_D subunitis unable to complex with the other RPA subunits. To examinethis, lysates prepared from cells transfected with the RPA2_wtand RPA2_D expression vectors were subjected to immunoprecipitationwith an anti-RPA1 antibody and immunoblotted for the presenceof RPA2. The two myc-RPA2 variants, as well as the endogenousRPA2, efficiently coprecipitated with the RPA1 subunit (Fig.3A, lanes 1 to 3). The RPA2_D protein was also found in the lysateat levels comparable to those of RPA2_wt, suggesting that thetwo proteins have similar stabilities (lanes 5 and 6). BecauseRPA1 and RPA2 complex formation requires the RPA3 subunit (24,44), these data indicate that the two mutants form RPA heterotrimerswith equivalent efficiencies.

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FIG. 3. RPA2_D-containing RPA heterotrimers are replication competent. (A) U2-OS cells were transfected with empty vector (lanes 1 and 4), myc-RPA2_wt (lanes 2 and 5), or myc-RPA2_D (lanes 3 and 6). Lysates were prepared from each batch of transfected cells, and the lysates were subjected to immunoprecipitation using anti-RPA1 antibodies. Immunoprecipitates (IP) (lanes 1 to 3) and aliquots of the lysates (lanes 4 to 6) were then analyzed for the presence of RPA2 by Western blotting analysis using RPA2 antibodies (which recognize both transfected and endogenous RPA2). (B and C) RPA heterotrimers that contained either RPA2_wt (lane 1) or RPA2_D (lane 2) were expressed in E. coli, purified, and analyzed by SDS-PAGE and Coomassie blue staining (B). The purified RPA was then assayed for the ability to support SV40 DNA replication in combination with an AS65 fraction purified from HeLa cells (51) (C). The open triangle shows that only background levels of DNA synthesis occur in reactions containing RPA2_wt but lacking T antigen. Similar results were observed using RPA2_D. (D) SV40 DNA replication reactions were performed in the presence of [ ${alpha}$ -³²P]dCTP to label the replication products as described in Materials and Methods. The reaction mixtures contained 600 ng of either RPA_RPA2wt (lanes 1, 2, 4, and 5) or RPA_RPA2D (lanes 3 and 6) and either lacked T antigen (lanes 1 and 4) or contained 750 ng of T antigen (lanes 2, 3, 5, and 6). After isolation, the DNA replication products were first linearized by restriction digestion and then either mock treated (lanes 1 to 3) or incubated with DpnI to cleave nonreplicated DNA (lanes 4 to 6). The digestion products were then subjected to agarose gel electrophoresis, and the images of the ethidium bromide (EtBr)-stained gel (to show the total level of DNA) and the autoradiograph of the gel (to visualize ³²P-labeled reaction products) are provided. The observed bands correspond to the linearized SV40 origin-containing plasmid.

To establish if heterotrimeric RPA containing the RPA2_D subunit(RPA_RPA2D) was inherently unable to function in DNA replication,we tested the abilities of RPA_RPA2D and RPA_RPA2wt to supportSV40 DNA replication in vitro. With the exception of the virallarge T antigen, SV40 replication is catalyzed by host cellcomponents (8, 22). The SV40 system can thus provide a relativelycomprehensive test of the ability of RPA to interact functionallywith ssDNA and the DNA replication machinery. Previous workby J. Hurwitz and colleagues has shown that separation of humancell extracts by ammonium sulfate precipitation yields two requiredfractions (AS30 and AS65), with RPA found to be the only essentialfactor within the AS30 fraction (51). Because the AS65 fractionlacks RPA, the activities of different RPA variants can be assayedby their abilities to complement the AS65 fraction in supportingSV40 DNA replication. The RPA_RPA2D and RPA_RPA2wt variants wereproduced in E. coli and purified to homogeneity (Fig. 3B). Useof the AS65 fraction alone showed no significant DNA replicationactivity (Fig. 3C). The addition of either heterotrimeric RPAcomplex supported T-antigen-dependent viral DNA replicationto similar extents, and the activities of the two RPA variantswere similar over a range of levels (Fig. 3C). The reactionproducts synthesized in the presence of RPA_RPA2D or RPA_RPA2wtwere equally resistant to DpnI, demonstrating that they werebona fide DNA replication products and not due to repair synthesis(Fig. 3D). RPA_RPA2D is therefore functionally active in supportingDNA replication in vitro. RPA_RPA2D was also found to bind normallyto short ssDNA oligonucleotides (4). These results are not completelyunexpected, as it was shown previously that the RPA phosphorylationstate does not appreciably affect the ability of RPA to functionin viral DNA replication or in DNA repair (2, 7, 36). In sum,mutation of the seven serines and one threonine in the N terminusof RPA2 to negatively charged aspartate residues does not haveany apparent effect on the inherent activity of the heterotrimericprotein.

We next examined the possibility that expression of the RPA2_Dmutant generates a signal that shuts down cellular DNA synthesisand thus indirectly prevents RPA2_D from associating with chromatin.To address this issue, cells were transfected with the RPA2_wtor RPA2_D expression construct and pulse-labeled with BrdU. Thecells were then subjected to FACS based on three signals: thelevel of myc-RPA2, DNA content, and BrdU incorporation. In additionto confirming that the two RPA2 variants were expressed at comparablelevels (Fig. 4A and C), it was found that the percentages ofcells in S phase were similar regardless of whether the cellswere transfected with RPA2_wt (Fig. 4B), RPA2_D (Fig. 4D), orempty vector (not shown). Although the percentage of cells inS phase was somewhat high compared to other experiments, perhapsbecause of transfection conditions, the fractions of cells inS phase were routinely found to be similar for RPA2_wt and RPA2_D.We conclude that expression of RPA2_D does not significantlyaffect cell cycle progression.

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FIG. 4. Expression of RPA2_D does not affect cell cycle progression. Cells transfected with myc-RPA2_wt (A and B) or myc-RPA2_D (C and D) were incubated with 10 µM BrdU for 30 min prior to harvest. The cells were then subjected to FACS analysis using a pairwise analysis of the levels of myc and BrdU signals. Transfected cells (with significant myc signals [boxed regions in panels A and C]) were further analyzed for the BrdU signal and the amount of DNA. (B and D) Fractions of cells in G₁, S, and G₂ phases. For each plot, the x and y axes indicate fluorescence intensities of the different signals.

RPA2_D is unable to complement the loss of endogenous RPA2. The data presented above suggested that the RPA2_wt subunit,but not RPA2_D, would be able to complement the loss of endogenousRPA2 and support chromosomal DNA replication. To test this possibility,cells were depleted of cellular RPA2 by using an siRNA moleculedirected against the 3' noncoding sequence of RPA2. The RPA2expression cassettes do not contain the siRNA-targeted sequences,and hence the myc-RPA2 RNA produced from these vehicles is resistantto siRNA-mediated degradation. Visualization of RPA2 in thesesiRNA-treated cells by epifluorescence microscopy showed anapparent reduction of the RPA2 signal to nearly background levelin >90% of the cells (compare Fig. 5D with A). Western blottinganalysis indicated that RPA2 levels were reduced by >95%(data not shown). Upon cotransfection with myc-RPA2_wt, a significantfraction of the cells demonstrated a robust myc signal boundto chromatin, with the pattern of binding similar to that seenin replicating cells (Fig. 5C). In contrast, little or no myc-RPA2_Dwas found associated with chromatin (Fig. 5F), even though comparablelevels of RPA2_wt and RPA2_D expression were detected in nonextractedcells (Fig. 5B and E, respectively). We therefore conclude thatRPA2_D, unlike RPA2_wt, is unable to complement the loss of endogenousRPA2 and support DNA replication. These data also indicate thatRPA_RPA2D is not prevented from binding ssDNA because of competitionwith the endogenous RPA but rather is inherently unable to productivelyinteract with the DNA replication machinery.

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FIG. 5. Lack of RPA2_D chromatin association in cells lacking endogenous RPA2. U2-OS cells were incubated with a control (i.e., scrambled) siRNA (A) or an siRNA specific for the 3' noncoding region of the RPA2 mRNA (B to F). The cells were simultaneously cotransfected with an empty control vector (D), myc-RPA2_wt (B and C), or myc-RPA2 _D (E and F). Forty-eight hours posttransfection, the cells were extracted with 0.5% (vol/vol) Triton X-100 for 5 min prior to formaldehyde fixation to reveal RPA associated with chromatin (C and F) or were fixed to show total endogenous or transfected RPA2 (A, B, D, and E). The cells were then stained with anti-RPA2 (A and D) or anti-myc (panels B, C, E, and F) antibody and then visualized by epifluorescence microscopy. Cells with representative signals were chosen.

RPA association with replication centers is dependent on the RPA2 N terminus negative charge. We next examined whether mutation of particular serine or threonineresidues to aspartate was responsible for the lack of RPA2_Dassociation with replication centers or whether it was a consequenceof the heightened negative charge at the RPA2 N terminus. Wefirst constructed serine-to-aspartate substitutions at the cyclin-cdk2sites S23 and S29 (RPA2_D2) (Fig. 6G). S29 is invariably modifiedin each form of phosphorylated RPA (53), and thus, the RPA2_D2mutant resembles the form found in the initial steps of theRPA phosphorylation pathway. Further intermediate RPA2 mutantswere designed to roughly follow the phosphorylation pathway,as suggested by the data of Zernik-Kobak and colleagues (53).However, it must be mentioned that the exact pathway of RPA2modification from the hypophosphorylated to the hyperphosphorylatedform is not known, and it is unlikely that a strict order ofmodification occurs in vivo. Additional serine-to-aspartatechanges were generated in the background of the RPA2_D2 mutant,with a total of three (RPA2_D3 and RPA2_D31), four (RPA2_D4 andRPA2_D41), or five (RPA2_D5) positions mutated. The sites mutatedin these RPA2 variants are also found to be modified in RPAwith an intermediate phosphorylation state in vivo.

Transfection of U2-OS cells indicated that all of the intermediateRPA2 mutants were expressed at similar levels (Fig. 6A and Band data not shown). Relative to RPA2_wt, the RPA2 mutants withtwo or three Ser $->$ Asp changes had two notable effects: (i) a modestlyreduced fraction of transfected cells showing mutant RPA2 boundto chromatin (Fig. 6H) and (ii) a reduction in the intensityof RPA2 bound to chromatin (see below). More dramatic effectswere observed when four or five serines were converted. ForRPA2_D41, the fraction of cells with significant chromatin bindingwas threefold less than for RPA2_wt, and this fraction was reducedto 8% for the RPA2_D5 mutant (Fig. 6H). The intensities of chromatinstaining for the intermediate RPA2 mutants were also greatlyreduced in individual cells, as demonstrated by comparing theaverage staining patterns of cells transfected with RPA2_wt andRPA_D41 (Fig. 6C and D, respectively [taken with identical exposuretimes]).

The decrease in association of RPA2 with replication centerswas most strongly correlated with the number of aspartate residuesrather than with changes at any particular positions. The notionthat the mutation of serines to aspartates per se (i.e., irrespectiveof the changes in the RPA2 negative charge) causes decreasedRPA binding to replication centers is argued against becausethe N terminus of RPA2 is not critical for DNA replication invitro for mammalian RPA (23) or in vivo for yeast RPA (38).These data therefore suggest that the increase in net negativecharge afforded by the increased number of aspartate residuesis the primary factor regulating RPA binding to chromatin. Althoughthe location of the aspartate residues did not appear to havemajor effects on RPA2 activity, we did note that mutation ofthe S33 site, known as a consensus sequence for PIKKs, appearedto have a somewhat more deleterious effect.

RPA2_D is recruited to DNA damage foci following genotoxic stress. Under DNA damage conditions, a significant change occurs inthe nuclear distribution of RPA, with the more diffuse punctatepattern seen during DNA replication transforming to bright,well-distinguished foci. In this state, RPA colocalizes witha number of repair and checkpoint proteins (e.g., ATR and Rad51)and is thought to demarcate the sites of DNA repair and/or unrepairablelesions (19, 20, 39, 54). Such stress conditions cause a subsetof the endogenous RPA pool to become hyperphosphorylated (seebelow). We therefore reexamined the behavior of RPA2_D and RPA2_Ain cells undergoing genotoxic stress.

Cells were transfected with the RPA2_wt, RPA2_A, or RPA2_D expressionconstruct and then treated with CPT. CPT inhibits topoisomeraseI, indirectly causing DNA double-strand breaks, and leads torapid and massive RPA phosphorylation (42). Similar to RPA2_wt(Fig. 7A to C), the RPA2_A variant colocalized with RPA1 in brightfoci following CPT treatment (Fig. 7G to I). Very similar fociwere observed for endogenous RPA2 (not shown). Thus, the phosphorylation-defectiveRPA2_A variant is apparently competent to bind chromatin bothin normal (above [Fig. 2L]) and in stressed cells.

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FIG. 7. RPA2_D binds chromatin and colocalizes with RPA1 after CPT treatment. U2-OS cells were transfected with myc-RPA2_wt (A to C), myc-RPA2_D (D to F), or myc-RPA2_A (G to I) vector. Forty-eight hours posttransfection, the cells were incubated with 1 µM CPT for 3 h, extracted with 0.5% (vol/vol) Triton X-100 for 5 min, fixed, and stained with anti-RPA1 (A, D, and G) and anti-myc (B, E, and H) antibodies. (C, F, and I) Colocalization of the two stains, enlarged from the boxed regions.

In sharp contrast to the inability of RPA2_D to stably associatewith replication centers, CPT treatment caused the RPA2_D variantto colocalize with RPA1 in DNA damage foci (Fig. 7D to F). Thenumber and distribution of these foci, as well as the intensityof staining, were indistinguishable from those observed withthe RPA2_wt (and RPA2_A) construct. Thus, although the RPA2_D mutantis unable to localize to replication centers, this defect doesnot extend to the involvement of RPA2_D in the DNA damage response.

We determined if the CPT-dependent recruitment of RPA2_D to DNAdamage foci was applicable to other stresses. We tested HU andaphidicolin, agents that do not directly cause DNA damage butrather result in stalling of the DNA replication fork. As cellswere incubated with HU from 1 to 3 h (Fig. 8E and F), a progressiveincrease in RPA2_D association with chromatin was observed, withmost cells demonstrating a dispersed staining pattern. A fractionof cells exhibited distinctive foci, and these showed significantcolocalization with ${gamma}$ -H2AX, the phosphorylated form of histonevariant H2AX that is a marker for sites of DNA damage (Fig.8I to L) (40). Similar effects of HU were noted on cells transfectedwith RPA2_wt (Fig. 8A and B). In contrast, treatment with aphidicolinfor 3 h did not stimulate RPA2_D association with chromatin (Fig.8M; data not shown), demonstrating reduced toxicity of aphidicolinrelative to HU under these conditions. Exposure to ionizingradiation (10 Gy) gave rise to staining patterns of RPA2_wt andRPA2_D similar to that found with CPT (data not shown).

In the functional absence of the budding yeast homologs of ATRand its downstream effector Chk1 (Mec1p and Rad53, respectively),replication forks have a greater propensity to collapse whenencountering DNA damage, yielding unregulated production oflong ssDNA regions (32, 43, 45). We therefore hypothesized thataddition of caffeine, an inhibitor of the ATR-ATM-dependentcheckpoint response (21, 41), to HU-treated cells would similarlylead to replication fork degradation. This in turn would causefaster induction of DNA damage foci and of RPA2_D localization.To test this hypothesis, RPA2_wt- or RPA2_D-transfected cellswere treated with HU for 1 or 3 h in the presence of caffeine.Particularly for RPA2_D, addition of caffeine dramatically increasedthe number and intensity of RPA2 foci at both the 1- and 3-htime points (Fig. 8G and H). Quantification of the effects onmyc-RPA2 localization demonstrated that caffeine greatly increasedthe fraction of HU-treated cells with significant RPA2_D andRPA2_wt signals (Fig. 8M).

The effects of these various stress conditions on endogenousRPA phosphorylation were also examined (Fig. 8N). Those stressconditions that resulted in significant RPA2_D chromatin bindingalso caused increased phosphorylation of endogenous RPA2, althoughCPT caused modification of a greater fraction of the RPA pool,as well as phosphorylation of more RPA2 sites, than HU. Enhancedphosphorylation of RPA following a 1-h treatment with HU andcaffeine was occasionally seen. Consistent with our resultsshowing the inability of aphidicolin to stimulate the chromatinbinding of RPA2_D, aphidicolin also did not induce RPA2 phosphorylation.Because caffeine has been demonstrated to be an inhibitor ofATM and ATR kinase activities (21, 41), the observed hyperphosphorylationof RPA probably results from the caffeine-insensitive activityof DNA-PK that is stimulated by collapsed replication forks.However, we note that a recent study found that caffeine caninhibit the checkpoint response without inhibiting ATR-ATM kinaseactivity in vivo (11), leaving open the possibility that thesekinases may still be responsible. In any case, these data indicatethat the rate and extent of RPA2_D (and RPA2_wt) localizationto sites of DNA damage correlate with the degree of DNA damagesustained during stress.

Localization of endogenous hyperphosphorylated RPA. The properties of endogenous hyperphosphorylated RPA were examinedusing an antibody generated against an RPA2 peptide doubly phosphorylatedon serine residues 4 and 8. Lysates prepared from untreatedor CPT-treated U2-OS cells were probed with either a generalRPA2 antibody or the pSer4/pSer8-RPA antibody (Fig. 9J). Thephosphospecific antibody selectively recognized a species fromCPT-treated cells that comigrated with hyperphosphorylated RPA2by Western blotting analysis. Prior incubation of the CPT-treatedlysates with phosphatase resulted in the loss of both the hyperphosphorylatedRPA2 form and reactivity by the phosphospecific antibody. Weconclude that the phosphospecific antibody recognizes a hyperphosphorylatedRPA2 species that is modified on Ser4 and Ser8.

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FIG. 9. Endogenous phosphorylated RPA (P-RPA2) does not localize to sites of DNA synthesis. (A to F) U2-OS cells were either mock treated (A and D) or treated with 2.5 mM HU for 3 h (B and E) or with 2.5 mM HU and 5 mM caffeine for 3 h (C and F). The cells were extracted to visualize the chromatin-bound form of RPA, fixed, and then stained either with anti-RPA2 (A to C) or anti-pSer4/pSer8-RPA2 (D to F) antibody. (G to I) U2-OS cells were either mock treated or treated with 1 µM CPT for 30 min, followed by an additional 2.5-h incubation in medium lacking CPT. The cells were incubated with 10 µM BrdU for 15 min prior to being processed. The cells were then extracted to visualize the chromatin-bound form of RPA, fixed, and stained either with anti-pSer4/pSer8-RPA2 (G) or anti-BrdU (H) antibody. (I) Merged staining pattern. (J) Extracts prepared from mock-treated or CPT-treated (1 µM for 3 h) cells were subjected to Western blotting (W) analyses using either anti-RPA2 (34A) monoclonal antibody (anti-RPA2) or a rabbit anti-pSer4/pSer8-RPA2 antibody (anti-P-RPA2). CPT-treated extracts were also incubated with ${lambda}$ protein phosphatase ( ${lambda}$ PPase), as indicated.

The phosphospecific antibodies were used to examine the localizationof the pSer4/pSer8 form of RPA in untreated U2-OS cells andin cells treated with HU alone or with HU and caffeine. In controlcells or in cells treated only with HU (Fig. 9D and E), littleif any pSer4/pSer8-RPA staining was detected. Following treatmentwith both HU and caffeine, the cells showed a dramatic increasein pSer4/pSer8-RPA staining (Fig. 9F). The staining patternwas nearly identical to that of total RPA2 (compare Fig. 9Cand F), and also showed good overlap with ${gamma}$ -H2AX staining (datanot shown). The colocalization of pSer4/pSer8-RPA with sitesof DNA synthesis was also examined. Cells were treated withCPT and then incubated with BrdU. The areas of pSer4/pSer8-RPAstaining did not colocalize with sites of remaining DNA synthesisto any significant degree (Fig. 9L). A majority of the RPA poolis hyperphosphorylated under these conditions (Fig. 8N), renderingsimilar experiments using general RPA2 antibodies uninformative.We conclude that the hyperphosphorylated form of RPA localizesonly to chromatin following DNA damage and is not significantlyassociated with sites of chromosomal DNA synthesis.

DISCUSSION

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Discussion

We find that the RPA2_D mutant that mimics the hyperphosphorylatedstate is prevented from stable association with replicationcenters in vivo. The lack of association with sites of DNA synthesisis also observed for endogenous hyperphosphorylated RPA andis not a result of competition with the nonphosphorylated protein.Importantly, the RPA_RPA2D protein has activity equivalent tothe wild-type protein both in ssDNA binding (4) and in SV40DNA replication in vitro. The inherent activity of hyperphosphorylatedRPA or RPA_RPA2D in vivo also appears normal because genotoxicstress causes these RPA species to localize to DNA damage focisimilarly to endogenous RPA2 and RPA2_wt. Our data thereforeindicate that the chromosomal DNA replication machine has theability to discriminate between RPA species with different phosphorylationstates. In addition to providing a means to regulate RPA loadingand hence DNA replication, RPA phosphorylation also has thepotential to mark sites of DNA damage or replication stressfor recruitment of repair factors.

Our data suggest a novel feature of eukaryotic DNA replication,namely, that RPA is actively loaded onto the ssDNA by the chromosomalreplication machinery. This model arises from the fact thatRPA_RPA2D, and by inference hyperphosphorylated RPA, is inherentlyactive in binding the ssDNA at a DNA replication fork but isunable to do so in vivo. The most logical explanation is that,as the duplex DNA is unwound by the advancing DNA helicase,the hypophosphorylated RPA is loaded onto the ssDNA by proteincomponents of the replication fork machinery. One could easilyenvision, for example, that the minichromosome maintenance (MCM)complex, suggested to be the eukaryotic replicative helicase(29) and known to interact with RPA (55), would load RPA moleculesin a step-by-step fashion as the ssDNA is generated. Selectivebinding of nonphosphorylated RPA (i.e., endogenous RPA, RPA_RPA2wt,or RPA_RPA2A) to MCM would therefore allow this RPA species tobind only to unwound DNA. (The MCM complex is not involved inSV40 DNA replication.) However, RPA interacts with various proteins,including the DNA polymerase ${alpha}$ -DNA primase complex (14), andRPA phosphorylation has been found to inhibit the associationwith DNA polymerase ${alpha}$ (34). Thus, discrimination of the RPA phosphorylationstate can be achieved by these or other replication factors.One alternative model that does not require concerted RPA loadingwould involve a discrimination filter that prevents access ofthe phosphorylated RPA to the replication fork. The nature ofsuch a filter would be difficult to envisage.

DNA-damaging stress relieves the inhibition of RPA_RPA2D chromatinbinding and causes RPA_RPA2D association with DNA damage foci,as evident by colocalization with ${gamma}$ -H2AX. That HU causes RPA_RPA2Dfoci to form and increases the level of RPA2 phosphorylationwhile aphidicolin does neither indicates that replication forkblockage is not sufficient for RPA_RPA2D chromatin binding butthat the presence of DNA damage or aberrant replication forkstructures is also required. This conclusion is strengthenedby our observation that inhibition of ATR- or ATM-mediated checkpointresponse by caffeine stimulates the rate of RPA associationwith DNA damage foci. Mutation of MEC1, the Saccharomyces cerevisiaeATR homolog, is known to cause the collapse of DNA replicationforks that have been stalled by treatment with HU or methylmethanesulfonate (32, 45), and such treatment leads to the productionof long ssDNA regions (43). Because of the high affinity ofRPA for ssDNA (27, 52), we propose that the increased availabilityof ssDNA releases the constraints on RPA loading seen duringnormal S-phase progression. Thus, under damage conditions, theRPA phosphorylation state no longer regulates the associationof RPA with chromatin.

Our data indicate that hyperphosphorylated RPA is preferentiallyassociated with sites of DNA damage. The specific associationof repair factors with this modified form of RPA would thereforeprovide a mechanism to recruit repair factors to sites of DNAdamage. Interestingly, the ATRIP-ATR complex has been foundto sense damaged DNA by recognition of RPA-ssDNA complexes.Clearly, RPA phosphorylation has the potential to regulate thebinding of ATRIP-ATR and thereby modify the cellular checkpointresponse. Although our examination of RPA2_D expression did notdetect any notable effects on cell cycle progression, it willbe interesting to examine whether RPA2_D and RPA2_A expressionin cells lacking endogenous RPA alters cellular proliferativecapacity or response to DNA damage.

Finally, our data indicate that hyperphosphorylation of RPAcan limit its ability to support chromosomal DNA replication.It is unlikely that this mechanism alone could cause significantreductions in the level of DNA synthesis during genotoxic stress.Under severe stress conditions, such as 1-h exposure to 1 µMCPT (Fig. 8N) or irradiation with 30 J of UV light/m² (53),the hyperphosphorylated form of RPA contributes $~$ 50% of the totalRPA pool prepared from asynchronous cells. Even though the fractionof hyperphosphorylated RPA may be higher in S-phase cells, thesedata suggest that enough hypophosphorylated RPA would be availableto sustain DNA replication. That being said, we and others havefound that stress conditions also lead to the inhibition ofRPA activity by other processes (9, 30, 48), including sequestrationof RPA by nucleolin (12, 47). Combined, these data suggest thatinhibition of RPA activity by multiple mechanisms can serveto repress chromosomal DNA replication following stress.

ACKNOWLEDGMENTS

We thank Kyung Kim and Diana Dimitrova for helpful discussionsduring the course of these experiments, Kristine Carta for experttechnical assistance, and John Hirsch for assistance with FACSanalysis.

J.A.B. was supported by NIH grant AI29963, DOD Breast CancerResearch Program DAMD17-03-1-0299, Philip Morris grant 15-B0001-42171,and the NYU Cancer Institute and the Rita J. and Stanley KaplanComprehensive Cancer Center (NCI P30CA16087). M.S.W. was supportedby NIH grant GM44721.

FOOTNOTES

* Corresponding author. Mailing address: Dept. of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, NY 10016. Phone: (212) 263-8453. Fax: (212) 263-8166. E-mail: james.borowiec{at}med.nyu.edu.

REFERENCES

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