Research Article
4909
A hypophosphorylated form of RPA34 is a specific component of pre-replication centers Patricia Françon1, Jean-Marc Lemaître1, Christine Dreyer2, Domenico Maiorano1, Olivier Cuvier1 and Marcel Méchali1,* 1Institute of Human Genetics, CNRS, Genome Dynamics and Development, 141, rue de la Cardonille, 2Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tûbingen, Germany
34396 Montpellier CEDEX 5, France
*Author for correspondence (e-mail:
[email protected])
Accepted 14 June 2004 Journal of Cell Science 117, 4909-4920 Published by The Company of Biologists 2004 doi:10.1242/jcs.01361
Summary Replication protein A (RPA) is a three subunit singlestranded DNA-binding protein required for DNA replication. In Xenopus, RPA assembles in nuclear foci that form before DNA synthesis, but their significance in the assembly of replication initiation complexes has been questioned. Here we show that the RPA34 regulatory subunit is dephosphorylated at the exit of mitosis and binds to chromatin at detergent-resistant replication foci that colocalize with the catalytic RPA70 subunit, at both the initiation and elongation stages of DNA replication. By contrast, the RPA34 phosphorylated form present at mitosis is not chromatin bound. We further demonstrate that RPA foci assemble on chromatin before initiation of DNA replication at sites functionally defined as initiation replication sites. Association of RPA with these sites does
Key words: Replication protein A, DNA replication foci, Xenopus, MCM, Nascent DNA, Aphidicolin
Introduction Replication protein A (RPA) is a stable complex of three different subunits (p70, p34 and p11) that participates in different cellular processes: DNA replication, recombination and repair (Wold, 1997). The RPA70 subunit has a high affinity for single-stranded DNA, but a DNA-binding activity that is associated with the RPA34 and RPA11 subunits (Bochkareva et al., 1998). Cell-cycle-regulated phosphorylation of RPA34 at the G1-S transition has been described (Din et al., 1990; Fang and Newport, 1993; Pan et al., 1994). However, it is not yet clear whether phosphorylation of RPA is involved in the onset of DNA replication (Pan et al., 1995; Philipova et al., 1996), as DNA replication efficiency is not affected by DNAdependent protein kinase (Brush et al., 1994; Lee and Kim, 1995; Pan et al., 1995) or Cdc2 (Henricksen and Wold, 1994), two kinases involved in RPA modification. The RPA70 large subunit alone is not sufficient for DNA replication, and the RPA complex cannot be replaced by E. coli single-stranded DNA-binding protein SSB (Dornreiter et al., 1992; Walter and Newport, 2000), suggesting that the RPA34 and RPA11 subunits are necessary for the function of RPA in DNA replication. RPA participates in the synthesis and processing of Okazaki fragments during DNA replication in viruses and in yeast (Mass et al., 1998; Bae et al., 2001). In multicellular organisms, however, its participation in the preinitiation complex is currently unclear. Notably, sites of DNA synthesis are detected as replication foci that co-localize with
RPA and the DNA polymerase δ processivity factor PCNA (Adachi and Laemmli, 1992; Dimitrova et al., 1999), but none of the proteins forming the pre-replication complex (e.g. ORC, cdc6, cdt1, MCMs) exhibits such clear localization. The significance of RPA foci is therefore debated. In Xenopus in vitro systems, RPA is present on chromatin before initiation of DNA replication. It first localizes at distinct foci that might be pre-replication centers, but appears to be relocalized evenly throughout the nucleus after initiation of DNA replication (Adachi and Laemmli, 1992). In mammalian cells, RPA localizes to replication foci at the onset of S phase (Brenot-Bosc et al., 1995; Murti et al., 1996), but RPA foci were not observed in early G1 (Dimitrova et al., 1999; Dimitrova and Gilbert, 2000). These observations have led to the proposal that RPA foci in Xenopus may be unrelated to initiation of DNA synthesis and may represent a nonspecific storage of RPA bound to chromatin (Dimitrova et al., 1999). We have investigated the association of the regulatory subunit RPA34 with chromatin during the cell cycle and its participation in pre-replication complexes. We show that the regulatory RPA34 subunit is rapidly dephosphorylated upon mitosis exit, and then associates with chromatin prior to the initiation of DNA replication. RPA34 is detected in its hypophosphorylated form during the whole of S phase and assembles into detergent-resistant foci. Mitosis results in phosphorylation of RPA34 and its loss of chromatin binding activity. At mitosis exit, RPA foci first form on sites that are
not require nuclear membrane formation, and is sensitive to the S-CDK inhibitor p21. We also provide evidence that RPA34 is present at initiation complexes formed in the absence of MCM3, but which contain MCM4. In such conditions, replication foci can form, and short RNAprimed nascent DNAs of discrete size are synthesized. These data show that in Xenopus, the hypophosphorylated form of RPA34 is a component of the pre-initiation complex. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/117/21/4909/DC1
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functionally defined as replication initiation sites. The assembly of these foci do not require nuclear membrane formation. We further show that RPA foci can assemble in MCM3-depleted extracts that still contain MCM4 bound to chromatin. Moreover, these RPA foci co-localize with stalled, short, nascent DNAs of discrete size.
37°C. DNA was then extracted with phenol/chloroform and further purified by gel filtration chromatography on a spin column (P6, BioRad). DNA was then incubated with 2 units of λ-exonuclease (Biolabs) for 4 hours at 37°C in a final volume of 40 µl. At the end of the incubation λ-exonuclease was inactivated by heating at 70°C for 15 minutes. Analysis of the products was done by electrophoresis on a 2% agarose gel.
Materials and Methods
Indirect immunofluorescence assays For analysis of chromatin-bound proteins, 10 or 15 µl samples were diluted 10 times with 0.3% Triton X-100 in XB and incubated for 5 minutes on ice. They were fixed by addition of an equal volume of 8% formaldehyde in XB, for 1 hour at 4°C. For analysis of total nuclear proteins, 10 µl samples were fixed with 200 µl of 4% formaldehyde in XB. Nuclei or chromatin were centrifuged onto glass coverslips at 1500 g for 8 minutes, through a 0.7 M sucrose cushion. Coverslips were post-fixed for 4 minutes in cold methanol and rehydrated for 15 minutes in PBS. After 1 hour saturation at room temperature in PBS, 1% bovine serum albumin (BSA), coverslips were incubated overnight at 4°C with specific antibodies. Each coverslip was washed 5 times for 20 minutes with 0.1% Tween-20 in PBS before incubation with secondary antibodies for 1 hour at room temperature, followed by five 20-minute washes with PBS, 0.1% Tween-20, and a 15 minute incubation in 10 µg/ml Hoechst 33258 in PBS.
Antibodies The anti RPA34-specific monoclonal antibody (324A.1) was isolated from a monoclonal antibody library raised against Xenopus oocyte germinal vesicle proteins (Dreyer et al., 1981) and recognizes only dephosphorylated RPA34 (Fig. S1, see supplementary material). The RPA polyclonal antibodies 309.112 and E-Ky were generous gifts of Y. Adachi (Institute of Cell and Molecular Biology, Edinburgh, UK). The 309.112 antibody recognizes both RPA70 and RPA34 (Adachi and Laemmli, 1994) but we observed that it recognized RPA34 only under its phosphorylated form (Fig. S1, see supplementary material). The E-Ky antibody is specific for the Xenopus RPA70 subunit (Adachi and Laemmli, 1992). Antibodies against Cdt1, MCM3 and MCM4 were previously described (Coué et al., 1996; Coué et al., 1998; Maiorano et al., 2000a). γ-H2AX polyclonal antibody was supplied by Interchim.
Xenopus egg extracts Interphase and mitotic (CSF) low-speed (LS) extracts were prepared according to protocols described in detail previously (Menut et al., 1999) and available at http://www.igh.cnrs.fr/equip/mechali/. Replication reactions Demembranated sperm nuclei were prepared as described (Menut et al., 1999). Nuclei were incubated in LS extracts (1000 nuclei/µl), or mitotic (CSF) extracts that were activated with 1 mM CaCl2. Extracts were supplemented with energy mix (10 µg/ml creatine kinase, 10 mM creatine phosphate, 1 mM ATP, 1 mM MgCl2), 150 µg/ml cycloheximide and reactions were incubated at room temperature, or 18°C for pulse assays. DNA synthesis was measured by [α32P]dCTP incorporation as previously described (Menut et al., 1999). DNA replication was also determined by immunofluorescence after 3minute pulses with 20 µM biotin-16-dUTP (Boehringer) at 18°C. Immunodepletions were performed as described (Maiorano et al., 2000a), except for MCM3 depletions, in which IgGs were coupled to protein-G Sepharose beads at 4°C. Purification and analysis of chromatin fractions 50 µl samples were diluted with 5 volumes of extract buffer (XB: 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM KOH-Hepes pH 7.7, 50 mM sucrose) and pelleted by centrifugation at 5000 g for 12 minutes through a 0.7 M sucrose cushion. After removing the supernatant, nuclear pellets were resuspended in XB, 0.3% Triton X100 and incubated for 5 minutes on ice. After a further 10,000 g centrifugation for 2 minutes, chromatin (pellet) and nucleosolic (supernatant) fractions were recovered. φX-174 ss or ds DNA incubated in Xenopus egg extracts was recovered after dilution with 5 volumes of XB, 1 mM ATP, 1 mM MgCl2, followed by gel filtration through a Sephacryl S-400 HR column (Pharmacia). λ-Exonuclease treatment of nascent DNA DNA purified from either mock-depleted or MCM3-depleted egg extracts was incubated with 0.4 mg/ml of proteinase K for 1 hour at
Microscopy and image analysis Confocal microscopy was performed using a Biorad 1024 CLSM system and a Zeiss LSM 510. Images were collected sequentially to avoid cross-contamination between fluorochromes. A series of optical sections were collected and projected onto a single image plane in the laser sharp 1024 software and processing system. Deconvolution imagery was performed on cut sections with a DMR Leica microscope coupled to a CCD Princeton Camera.
Results Two distinct populations of the regulatory RPA34 subunit are present at mitosis and interphase with opposite chromatin binding activity Xenopus egg extracts mimic most events occurring during S phase and mitosis and are particularly suitable for biochemical analysis of initiation of DNA replication. Xenopus metaphasearrested egg extracts were supplemented with sperm chromatin and released into interphase by calcium addition. Fig. 1A shows the onset of S phase, whereas RPA modifications and binding to chromatin were analyzed during mitosis exit and S phase in Fig. 1B. We used two different antibodies that recognize two RPA34 populations. A polyclonal antibody raised against the RPA complex present in mitotic extracts (Adachi and Laemmli, 1994) (see also Materials and Methods) recognizes both RPA70 and RPA34. This antibody recognized RPA34 only in its hyperphosphorylated form (Fig. S1, see supplementary material). A monoclonal antibody isolated from a library of monoclonal antibodies raised against Xenopus oocyte germinal vesicle proteins (Dreyer et al., 1981) recognizes the RPA34 subunit only in its hypophosphorylated form (Fig. S1, see supplementary material for a detailed characterization). Nuclei were purified by low-speed centrifugation, and detergent-extracted to obtain nucleoplasmic (S) and chromatin (Chr) fractions. The fractions were run onto an SDS-PAGE,
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Fig. 1. RPA34 is present as a hypophosphorylated form during the entire S phase. (A) Demembrated sperm nuclei (1000/µl) were incubated in a mitotic extract to which calcium was added to promote entry in S phase. DNA synthesis was followed by incorporation of [α-32P]dCTP. (B) Nuclei were isolated and treated with 0.3% Triton X-100, to collect the chromatin-bound (Chr) and nucleosolic-unbound (S) fractions, as described in Materials and Methods. The 309.112 polyclonal antibody (pAb) was used to reveal the RPA70 and RPA34, while dephosphorylated RPA34 was detected with the monoclonal antibody (mAb), as described in Materials and Methods and Fig. S1 (see supplementary material). (C) The binding of RPA to chromatin was analyzed during DNA replication and entry in mitosis induced by addition of 30 µg/ml of nondegradable B cyclin (cyclin B∆90). Nuclei were isolated and treated with 0.3% Triton X-100 to collect the chromatin-bound (Chr) and -unbound (S) nuclear fractions, as described in Materials and Methods. In mitotic or mitotic like-extracts (+ ∆ cyclin), the nuclear envelope does not form and the chromatin-associated (Chr) and cytoplasmic (Cyto) forms of RPA were analyzed. Fractions were analyzed by 12.5% SDS-PAGE and immunoblotted either with the 309.112 polyclonal antibody (pAb), or the monoclonal antibody (mAb) specific for dephosphorylated RPA34.
blotted and RPA was detected using the two specific RPA antibodies. At mitosis, RPA34 is phosphorylated and does not bind to chromatin (Fig. 1B,C). When exit from mitosis is induced, RPA34 is dephosphorylated before entry into S phase (Fig. 1B, mAb, 30 minutes). Dephosphorylated RPA34 starts to accumulate on chromatin (Chr, 30-60 minutes), where it remains throughout S phase. The RPA70 subunit shows the same behaviour; it does not associate with chromatin at mitosis but does during S phase. During a normal cell cycle, we never observed the simultaneous presence of the two RPA34 hyperphosphorylated and hypophosphorylated populations. Fig. 1C suggests that hyperphosphorylation of RPA at mitosis is under the control of cdc2-cyclin B, as addition of a non-degradable form of cyclin B to the interphase extract was sufficient to induce a complete disappearance of the S-phase-specific hypophosphorylated form. Concomitantly to the hyperphosphorylation of RPA34, the chromatin binding activity of the complex was lost. The S-phase-specific RPA34 isoform co-localizes with RPA70 and DNA replication sites at both the initiation and elongation stages of DNA replication RPA foci co-localize with replication sites during S phase, both in Xenopus (Adachi and Laemmli, 1992; Adachi and Laemmli, 1994) and in mammals (Dimitrova and Berezney, 2002). However, in Xenopus egg extracts, RPA foci appear to redistribute uniformly in the nucleus during S phase (Adachi and Laemmli, 1992; Yan and Newport, 1995). This was suggested to be the consequence of the redistribution of RPA along single-stranded DNA during the elongation stage of replication. RPA foci have also been detected in ORC- and Cdc6-depleted extracts (Coleman et al., 1996), but these experiments were performed using membrane-depleted
Xenopus egg extracts, which are not competent to initiate DNA replication. Contradictory results, both for the nuclear distribution as well as for the interaction between RPA34 and RPA70, have also been reported (Cardoso et al., 1993; Brenot-Bosc et al., 1995; Murti et al., 1996; Treuner et al., 1999). We first asked whether the hypophosphorylated RPA34 form present in S phase also assembles into nuclear foci with RPA70. To perform this study, we used the corresponding monoclonal antibody described above, and a polyclonal antibody specific for RPA70 (Adachi and Laemmli, 1992). Fig. 2 shows that RPA34 and RPA70 subunits are first localized in foci before complete nuclear membrane formation (30 minutes) and before initiation of DNA replication. A uniform distribution of RPA was then observed during S phase (60 minutes, Fig. 2A). However, when nuclei were treated before fixation with Triton X-100, which removes the nuclear membrane and nucleoplasmic fraction, RPA34 and RPA70 subunits were always found in nuclear foci, most of which colocalize throughout S phase (Fig. 2B). We conclude that both subunits RPA70 and RPA34 co-localized before initiation of DNA replication, as well as during initiation and elongation of DNA synthesis. Our observations on intact or detergent-treated nuclei confirm biochemical data of Fig. 1 showing that RPA is present as two fractions in the nucleus, one bound to chromatin and the other unbound. The increase in nuclear staining of RPA during S phase that we and others have observed (Adachi and Laemmli, 1992; Yan and Newport, 1995) was due to the unbound form that accumulates in the nucleus. By contrast, the level of chromatin-bound RPA remains constant and present in foci that contain both the hypophosphorylated form of the regulatory RPA34 subunit and the catalytic RPA70 subunit, throughout S phase. To further confirm that RPA34 foci are active replication
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Fig. 2. Co-localization of RPA34 subunit with RPA70 and DNA replication foci. (A) Sperm nuclei were incubated in calcium-activated egg extracts and samples were fixed 30 and 60 minutes later and analyzed by fluorescence microscopy. DNA was detected using Hoechst (Aa,d). The RPA34 subunit was detected using the specific monoclonal antibody and a Texas Red-conjugated anti-mouse antibody (Ab,e). The RPA70 subunit was detected using the specific polyclonal E-Ky antibody (Materials and Methods) and a FITC-conjugated anti-rabbit antibody (Ac,f). (B) Nuclei were treated with 0.3% Triton X-100 before formaldehyde fixation at the indicated times, and the analysis was carried out using confocal microscopy. RPA34 (Ba-d) and RPA70 (Be-h) were visualized as in panel A. The overlap of RPA34 and RPA70 signals is shown (merge). Bar, 5 µM. (C) Sperm nuclei were incubated in egg extracts, and samples were pulse labeled for 3 minutes with biotinylated nucleotides. Three different nuclei are shown. RPA34 was detected using the monoclonal antibody and a secondary FITC-conjugated antimouse antibody. Nucleotide incorporation was visualized with Texas Red-conjugated streptavidin. Analysis was performed by deconvolution and the merge of the two signals is shown (merge, yellow). Bar, 5 µM.
sites, nuclei were pulse-labeled with biotin-dUTP and then incubated with the RPA34-specific monoclonal antibody. Single cut sections of nuclei, analyzed by deconvolution microscopy, demonstrate that most biotin-dUTP foci (Fig. 2, replication foci, in red) co-localized with RPA34 foci (Fig. 2C). We also observed RPA34 foci (Fig. 2, green) that were not yet engaged in DNA replication, since DNA replication within nuclei is not perfectly synchronous. The formation of these foci is sensitive to p21 (Cip1), an inhibitor of cdk2-cyclin E that inhibits the initiation of DNA replication by blocking the unwinding step (Fig. 3A,B) (Walter and Newport, 2000; Adachi and Laemmli, 1994; Jackson, 1995; Yan and Newport, 1995). Finally, we also detected the hypophosphorylated RPA foci when sperm chromatin is assembled in Xenopus high-speed interphase egg extracts (HSE). These extracts cannot form nuclear membranes and, therefore, do not permit replication (Blow and Laskey, 1986; Mechali and Harland, 1982). The RPA foci observed do not appear to be related to DNA repair since γ-H2AX foci, which localize at sites of DNA damage (Kobayashi et al., 2002; Furuta et al., 2003) (Fig. S2, see supplementary material), were not detected (Fig. 3C). The formation of RPA foci in HSE is consistent with the early binding of RPA34 to chromatin (within 15 minutes), before nuclear membrane formation and the initiation of DNA
replication in low-speed egg extracts (Fig. 1) (Adachi and Laemmli, 1994; Coué et al., 1996). RPA assemble at pre-replication foci The detection of RPA in discrete chromatin foci at the transition between formation of pre-RCs and the initiation of DNA synthesis is in agreement with its proposed function as a singlestranded DNA-binding protein required to unwind DNA at replication origins. However, in mammalian cells it has been suggested that RPA foci observed before initiation of DNA synthesis are not in nuclear structures involved in initiation of DNA replication (Dimitrova et al., 1999). As such, it is not yet clear whether RPA foci correspond to future replication initiation foci, where DNA replication starts. To address this question it is important first to identify what are the future replication initiation foci. We therefore designed an experiment in which we could follow the assembly of RPA on sites already identified as replication initiation sites. We first labeled replication initiation sites on sperm chromatin incubated in Xenopus egg extracts in the presence of biotin dUTP and aphidicolin. The aphidicolin block was then released to allow S phase to proceed, leading to sperm chromatin containing replication initiation sites functionally tagged. The scheme of
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Fig. 3. RPA34 foci do not form in the presence of p21 but assemble in the absence of nuclear membrane formation. (A) Sperm nuclei were incubated in a calcium-activated mock (u) or p21-treated (e) extract. DNA replication was monitored by incorporation of [α32P]dCTP (Materials and Methods). (B) The same experiment as in A except that biotin-dUTP was used to follow DNA synthesis. Samples were treated with 0.3% Triton X-100 before formaldehyde fixation, and immunofluorescence analysis was carried out with the RPA34 monoclonal antibody at the initiation (30 min) or elongation stage (60 min) of DNA replication. (C) Sperm nuclei were incubated in low-speed egg extracts (LSE) or high-speed egg extracts (HSE) as above. DNA was stained with Hoechst, RPA34 was detected using the monoclonal antibody and γ-H2AX, a marker of DNA damage, with a specific polyclonal antibody (see Materials and Methods; Fig. S2, see supplementary material).
the experiment is outlined in Fig. 4A. We then asked whether, in the following cell cycle, RPA assembles at these sites that are functionally defined as DNA replication foci, or randomly on chromatin, before initiation of DNA synthesis. As expected, during the first cell cycle, most RPA foci observed on chromatin co-localized with replication foci labeled with biotin dUTP (Fig. 4B, columns 1 and 2). At the end of S phase, as determined by introduction of bio-dUTP during the elongation phase (Fig. S3, see supplementary material), recombinant cyclin B∆90 was added to induce mitosis. RPA was concomitantly released from chromatin (Fig. 4B, column 3), while the previously labeled replication initiation foci were still detected on condensed chromatin. We then monitored the re-binding of RPA at 5 minute intervals, immediately after entry into a new interphase, which was induced by calcium. Strikingly, most RPA foci that reformed on chromatin were found to assemble on replication initiation sites functionally tagged during the previous cell cycle (Fig. 4B, column 4), demonstrating that RPA associates with chromatin sites that are replication initiation foci. To confirm these data, we used a similar protocol, except that at the end of the first cell cycle nuclei were transferred to a membrane-depleted egg extract (Fig. 5, high-speed extract, HSE) in which DNA synthesis on sperm chromatin does not
occur (Mechali and Harland, 1982; Blow and Laskey, 1986) but pre-RCs are formed (Coué et al., 1996; Walter et al., 1998). We labeled replication initiation foci during a short period of time in the first cell cycle to ask whether RPA assembles on all of them during the second cell cycle. We also checked for the absence of DNA synthesis during the nuclear transfer in the high-speed extract (data not shown), by addition of a second dUTP analog (digoxigenin dUTP). The results of this experiment (Fig. 5) show that, in high-speed egg extracts, RPA assembles on chromatin into foci that co-localize with the replication initiation foci previously identified. Some additional RPA foci were also detected that may represent replication initiation sites that were not labeled during the first cell cycle in the protocol used. Together these experiments show that RPA foci, which form on chromatin before initiation of DNA synthesis, are structures functionally related to DNA synthesis. MCM3-depleted extracts assemble MCM4 and RPA onto chromatin, and initiate the synthesis of short nascent DNAs DNA replication initiation occurs through a multistep process
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involving the assembly of a pre-replication complex, and then the recruitment of the MCM helicase complex (Takisawa et al., 2000; Lei and Tye, 2001). In Xenopus, the assembly of RPA foci was previously shown to be regulated by FFA-1, a homologue of Werner helicase (Yan et al., 1998). However,
recent data (Chen et al., 2001) report that immunodepletion of FFA-1 neither inhibits DNA replication nor impairs the assembly of RPA foci. To characterize the RPA contribution to the unwinding reaction, we have taken advantage of an antiMCM3 monoclonal antibody that completely removes MCM3associated MCM2-7 proteins from egg extracts (Maiorano et al., 2000a). However, a discrete fraction of MCM4 remains present even after a second round of MCM3 depletion (Fig. 6A), in agreement with the presence of different MCM subcomplexes (Thommes et al., 1997; Coué et al., 1998; Maiorano et al., 2000a; Mendez and Stillman, 2000; Prokhorova and Blow, 2000). DNA replication was blocked in the MCM3-depleted extracts (Fig. 6B), and we confirmed the absence of MCM3 but the presence of MCM4 bound to chromatin by immunofluorescence analysis of nuclei (Fig. 6C). In the MCM3-depleted extract, staining of chromatin with the RPA34-specific monoclonal antibody shows that hypophosphorylated RPA34 assembles into foci (Fig. 6D). These results show that inhibition of the assembly of the whole MCM2-7 helicase complex on chromatin does not prevent the formation of RPA foci. Although the presence of the whole MCM complex is necessary for an efficient and processive helicase activity, MCM4 appears as a crucial component of the helicase activity (Zou and Stillman, 2000; Ishimi et al., 2003), and is the closest homologue to the unique MCM subunit found in Archaebacteria (Kearsey and Labib, 1998; Kelman et al., 1999; Chong et al., 2000). This led us to consider the possibility that pre-licensed origins containing both MCM4 and RPA34 could permit limited DNA synthesis at origins. As shown in Fig. 6D, nuclei formed in MCM3-depleted extracts show significant incorporation of the nucleotide analog biotin-dUTP. Moreover, the biotin-dUTP labeling was not evenly distributed in the nucleus, but appeared as distinct foci that co-localized with RPA34 foci. To investigate the nature of the limited DNA synthesis observed in MCM3-depleted extracts, we analysed the length of DNA synthesized by alkali agarose gel electrophoresis allowing the separation of both high molecular weight and low molecular weight products. Synthesis due to DNA repair should lead to Fig. 4. RPA assembles to pre-replication foci. (A) Experimental scheme. the appearance of labeled DNA of high molecular Sperm chromatin was incubated for 60 minutes at 22°C in a low-speed egg weight, whereas nascent DNA synthesized at origins extract (LSE) in the presence of aphidicolin (5 µg/ml) to slow down DNA should lead to the accumulation of labeled DNA of replication (95% inhibition of total nucleotide incorporation, data not shown) short and discrete size. Fig. 7A reveals that DNA and to label replication initiation foci with biotinylated dUTP. After a first synthesized in MCM3-depleted extracts is mainly of wash, fresh LSE was added and elongation allowed to proceed for 60 minutes low molecular weight, in a distinct 150-350 bp range. without biotin dUTP. Then purified recombinant cyclin B∆90 was added to Importantly, this discrete population of short nascent induce mitosis. After a second wash, calcium was added to promote the exit from mitosis and entry in a new S phase cycle. (B) Nuclei were analyzed by DNAs was not observed in mock-depleted extracts. It immunofluorescence at each step of the reaction to detect DNA (DAPI is not detected in extracts depleted of the Cdt1 protein staining), RPA (monoclonal antibody) and dUTP incorporation. The merged (Fig. 7B), which is absolutely required for the loading images of RPA and dUTP are also shown. DNA damage was analyzed using of MCM2-7 complexes at origins of DNA replication an γ-H2AX antibody, a known marker of DNA repair (Furuta et al., 2003; (Maiorano et al., 2000b; Nishitani et al., 2000; Kobayashi et al., 2002) (Fig. S2, see supplementary material). In row 2, Wohlschlegel et al., 2000). elongation was without biotin-dUTP, but it was also followed by adding We conclude first that in the absence of MCM3, biotin-dUTP in a sample resulting in a homogenous dUTP staining (Fig. S3, chromatin containing MCM4 exhibits limited and see supplementary material). In row 4, the reassembly of RPA was Cdt1-dependent DNA synthesis and, secondly, that monitored at 5 minute intervals and occurred 10 to 15 minutes after adding nascent DNA co-localizes with RPA34 foci. fresh LSE.
RPA is a component of Pre-RCs Two stages in the formation of initiation complexes containing RPA are revealed by uncoupling DNA helicase from the DNA synthesis machinery We further asked to what extent nascent DNA formed in MCM3depleted extracts compares with synthesis in the presence of aphidicolin, which does not inhibit the assembly of the MCM27 helicase complex but inhibits DNA polymerase-α. The analysis was performed at early time points following the aphidicolin block, immediately after nuclear membrane formation, to avoid potential artefacts induced by re-initiation events, and samples were analyzed by urea-polyacrylamide gel electrophoresis to increase the resolution of low molecular weight products. The population of short DNA synthesized in the MCM3-depleted extracts was resolved into two populations of 120-160 nt and 200-320 nt (Fig. 8A). By contrast, in aphidicolin-treated samples, a discrete product estimated to be 39 nt (arrow) was observed in addition to a broad population of up to 300-500 nt DNA products. The 39 nt nascent strand population progressively disappears during prolonged incubations with aphidicolin, at the expense of higher molecular weight species that slowly escape the aphidicolin block, leading
Fig. 5. RPA reassembles to pre-replication foci in high-speed egg extracts. (A) Experimental scheme. Sperm nuclei were incubated as described in legend to Fig. 4 except that cycle 2 was followed both in a low-speed egg extract (LSE) and a high-speed egg extract (HSE). (B) The assembly of RPA34 was analyzed at the entry in a new interphase in LSE or HSE, as in legend to Fig. 4.
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to the formation of 100-500 nt nascent strands. The 39 nt product resulting from the aphidicolin treatment is also degraded by DNase (Fig. 8B) and resistant to RNase treatment although its size is slightly decreased, suggesting a partial degradation of the RNA primers. Such short nascent DNA products synthesized in the presence of aphidicolin have been detected during SV40 DNA replication (Anderson and DePamphilis, 1979; Decker et al., 1986; Nethanel and Kaufmann, 1990) but so far have not been reported for genomic DNA replication. Fig. 8B shows that the two nascent strand populations, at 120160 nt and 200-320 nt, observed in MCM3-depleted extracts, were degraded by DNase I but not RNase. To further characterize the intermediates we repeated the experiment and assayed for the presence of RNA primers at the 5′ end of the nascent DNAs. λ-Exonuclease, an enzyme that degrades DNA from its 5′ end except when RNA primers are present, has been extensively used for isolation of nascent DNA at origins of DNA replication (Bielinsky and Gerbi, 1998; Abdurashidova et al., 2000). Fig. 8C shows the two nascent strand populations that accumulate only in MCM3-depleted extracts. Addition of λexonuclease results in the degradation of the upper band but not the lower band. This result suggests that the 120-160 bp corresponds to an RNA-primed nascent DNA. Its length is in full agreement with the observation that the first two nascent DNA fragments initiated at the human lamin B2 origin are close to 140 bp (Abdurashidova et al., 2000) and that the major nascent DNA products in yeast are 125 bp (Bielinsky and Gerbi, 1999). The upper band is likely to be the result of the first two successive nascent DNAs linked together. RNA primers are removed from these nascent DNA that accumulate when DNA synthesis is arrested by the lack of the processive MCM helicase. These data define two discrete intermediate states in the progression of the pre-RC to the initiation complex. A population of replication initiation intermediates of 39 nucleotides in length is synthesized by the aphidicolin-resistant DNA polymerase-α-primase activity when DNA synthesis is blocked by aphidicolin, whereas the DNA helicase remains active. A second population of replication initiation intermediates is revealed when the fully processive MCM2-7 helicase activity is inhibited. DNA synthesis starts with synthesis of an RNA-primed DNA of 125 bp but it becomes rapidly arrested, and is limited to the first 300 bp, probably soon after synthesis of the next nascent DNA. The RPA34 hypophosphorylated form is present at both kinds of replication initiation intermediates. Discussion Two RPA34 populations during the cell cycle with opposite chromatin binding activity In this paper we provide new insight into the nature and function of the foci to which RPA localizes during DNA replication. RPA34 cycles between two forms during cell division. Phosphorylated RPA34 is present at mitosis as previously observed both in Xenopus (Fang and Newport, 1993) and yeast (Din et al., 1990), but hypophosphorylated RPA34 is the only population detected from the exit of mitosis to the end of S phase, and it is present during both the initiation and elongation stage of unperturbed DNA replication. The induction of phosphorylation by a non-degradable form of cyclin B as well as the absence of phosphorylation when
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Fig. 6. RPA foci assemble in MCM3-depleted extracts. (A) Western blot of Xenopus egg supernatants (S1, S2) and protein precipitates (P) resulting from a single (S1) or double (S2) immunodepletion with either control mouse IgG (mock) or an anti-MCM3 monoclonal antibody (Materials and Methods). (B) Sperm nuclei were incubated in mock (u) or MCM3-depleted extracts (s) for different times. DNA replication was followed by incorporation of [α32P]dCTP. (C,D) Sperm nuclei were incubated in mock- or MCM3-depleted extracts, in the presence of biotin dUTP, as described in Materials and Methods. Samples were treated with 0.3% Triton X-100 before formaldehyde fixation, for the analysis of chromatin-bound proteins by immunofluorescence with antibodies specific for the indicated proteins.
synthesis of cyclin B is inhibited by cycloheximide (data not shown) is in agreement with previous observations showing that cdc2-cyclin B is responsible for the hyperphosphorylation of RPA at mitosis (Fang and Newport, 1993). The hyperphosphorylated mitotic form of RPA34 does not bind chromatin, abruptly disappears at exit from mitosis, and is replaced by a hypophosphorylated form. This is in complete agreement with recent data showing that phosphorylation of RPA34 prevents association with replication centers (Vassin et al., 2004). We also observed inhibition of RPA34 dephosphorylation by okadaic acid (data not shown), which suggests that phosphatase 1A/2A is involved in this dephosphorylation. This is in agreement with the inhibition of DNA replication observed after immunodepletion of phosphatase 2A from Xenopus egg extracts (Lin et al., 1998). We find that hypophosphorylated RPA34 is the only isoform present from the exit of mitosis to the end of S phase; it binds rapidly to chromatin and assembles in discrete nuclear foci, before initiation of DNA synthesis and during the entire process of DNA replication. Both dephosphorylated RPA34 and RPA70 co-localize with replication foci throughout S phase. The homogeneous staining previously observed in S phase nuclei was due to nucleoplasmic staining and does not reflect the chromatin-bound RPA population.
Association of RPA with pre-replication complexes Upon incubation of sperm nuclei in egg extracts, clear RPA foci appear after 10-20 minutes (Adachi and Laemmli, 1992; Coué et al., 1996), which is before formation of a nuclear membrane, whereas DNA synthesis starts at 40 minutes, with a delay that corresponds to the formation of the nuclear membrane and the recruitment of cdc45-DNA polymerase-α complex onto chromatin (Mimura and Takisawa, 1998). In S. cerevisiae, as well as in mammalian cells, RPA does not associate with chromatin before the onset of S phase (Tanaka and Nasmyth, 1998; Dimitrova et al., 1999; Zou and Stillman, 2000). These differences may simply reflect different kinetics in the formation of replication complexes. In Xenopus egg extracts, all proteins required for DNA replication are already present in an active form in the extract. The RPA binding chromatin step might take place earlier, before nuclear membrane formation, and consequently be detected as an intermediate step, as DNA synthesis will not proceed before the nuclear membrane is formed. Our data also show that the RPA foci that were previously observed in high-speed egg extracts (Adachi and Laemmli, 1992) represent replication-related foci. RPA assembles to replication initiation foci during the S phase, disassembles from chromatin during mitosis, and reassembles to the same foci during the second cell cycle, before DNA synthesis is initiated.
RPA is a component of Pre-RCs
Such association suggests that the organization of chromosomal regions for DNA replication is more conserved than expected, even in conditions where sequence-specific origins are not detected (Laskey and Harland, 1982; Mechali and Kearsey, 1984; Hyrien et al., 1995). Up to 200-300 replication initiation foci are formed during S phase in an egg extract (Coué et al., 1996; Mills et al., 1989), a value not so different from that of somatic cells (reviewed by Jackson and Pombo, 1998; Dimitrova et al., 1999; Berezney et al., 2000). As each of the foci contains several clustered replication origins (reviewed by Berezney et al., 2000), our results suggest that the specificity of the organization of replication domains in replication foci does not depend on the precise location of the origins in each replicon. If RPA foci are pre-replication foci, why have RPA foci been detected in extracts that have been depleted of ORCs with a specific antibody (Coleman et al., 1996)? At present, we envisage two main possibilities: first, ORC proteins are in large excess in the egg extracts (Rowles et al., 1996), and a small amount of ORC (Walter and Newport, 1997) is sufficient to allow formation of replication complexes. A second possibility is that ORCs are not necessary for RPA binding to DNA replication origins because RPA does not bind to chromatin at the same DNA element recruiting ORC. Both steps, although apparently independent, might still be involved in the formation of a larger initiation complex. This hypothesis is compatible with our observation that RPA is recruited to foci that are replication initiation foci, and recent data suggesting that origins in multicellular eukaryotes are composite structures involving easily unwound regions (Anglana et al., 2003; Kong et al., 2003). Another observation in agreement with this possibility is the inhibition of the formation of RPA foci by p21. This cdk2-cyclin E inhibitor prevents the entry of cdc45 and DNA polymerase-α, and consequently initiation of DNA synthesis, but allows formation of pre-replication complexes consisting of ORC, cdc6 and MCM (Yan and Newport, 1995; Hua and Newport, 1998; Mimura and Takisawa, 1998). Our favored model is that RPA foci would be structures distinct from pre-RC, formed without requiring pre-
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Fig. 7. Short nascent DNAs are synthesized in MCM3-depleted extracts. Alkaline agarose gel electrophoresis of DNA synthesized in egg extracts depleted with control mouse IgGs (mock-depleted), anti-MCM3 (MCM3-depleted) (A) or Cdt1 (Cdt1-depleted) antibodies (B). Depletion of the Cdt1 protein was over 98% (data not shown).
RC proteins, which will be nevertheless used later for setting the replication initiation centers. It has been previously reported that RPA binding to chromatin occurs after the Cdc45 binding step (Mimura et al., 2000; Walter and Newport, 2000), a result that apparently contrasts with other observations (Adachi and Laemmli, 1992; Coleman et al., 1996) (this study). However, Mimura et al. have shown that RPA binds to chromatin before Cdc45, but less tightly than during initiation of DNA synthesis (Mimura et al., 2000). They proposed that cdc45 is required for the unwinding of DNA, which in turn leads to the tight binding of RPA to the single-stranded DNA unwound at the replication origins. These data, together with our results, are in agreement with two binding modes of RPA, before and after initiation of DNA synthesis, with the association of RPA to chromatin stabilized at initiation of DNA replication through DNA-unwinding at the origin. The strength of RPA binding to chromatin would be a result of the extent of single-stranded DNA generated at the initiation and elongation steps of DNA synthesis so that RPA would be much more tightly associated with chromatin during elongation than during initiation. In another report (Walter and Newport, 2000), the binding of RPA to chromatin was challenged with high concentrations of detergent, which probably removed the less tightly bound RPA population in high-speed extract. However, a nucleoplasmic egg extract that neither depends on nuclear membrane to initiate DNA synthesis nor forms nuclei was also used in these experiments, which could lead to different results. Short nascent DNAs at initiation of DNA synthesis We showed that in MCM3-depleted extracts, in which the processive MCM2-7 helicase complex is inhibited, limited DNA synthesis is observed. RPA34 localizes under its
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Fig. 8. Discrete sizes of nascent DNAs in the absence of MCM3 or during inhibition of DNA replication by aphidicolin. The DNA replication products of MCM3-depleted extracts, or extracts treated with 15 µg/ml aphidicolin, were resolved by denaturing 8 M urea 6% acrylamide gel electrophoresis. Untreated samples (–), treated for 30 minutes at 37°C with 0.1 mg/ml RNase A (R), or with 1.3 U/ml DNase I, are shown for both MCM3depleted or aphidicolin blocked extracts. (B) DNA replication was followed by [α32P]dCTP incorporation. (C) The labeled DNA replication products from mockdepleted or MCM3-depleted egg extracts were separated by alkaline 2% agarose gel electrophoresis, which provides less resolution than acrylamide gel electrophoresis but gives better separation of the two populations of nascent DNAs. Incubation with (+) or without (–) λexonuclease (λ-exo) is shown.
hypophosphorylated form to foci at which short nascent DNA strands are synthesized, which is also the case during an unperturbed S phase. Moreover, this abortive DNA synthesis is aphidicolin-sensitive (data not shown). The association of MCM4 with chromatin in the absence of MCM3 has been observed both in Xenopus and human Hela cells (Coué et al., 1998; Maiorano et al., 2000a; Mendez and Stillman, 2000), and may represent a first stable intermediate in the loading of the full MCM2-7 helicase complex. MCM3, MCM5 and MCM4-7 subcomplexes have been documented (Burkhart et al., 1995; Ishimi et al., 1996; Schulte et al., 1996; Ishimi, 1997; Thommes et al., 1997; Coué et al., 1998; Sherman and Forsburg, 1998; Maiorano et al., 2000a; Prokhorova and Blow, 2000; Lee and Hurwitz, 2001), and a low helicase activity is associated only with the MCM4-7 complex in eukaryotes (Ishimi et al., 1996; Ishimi, 1997; You et al., 1999; Ishimi and Komamura-Kohno, 2001; Lee and Hurwitz, 2001). Interestingly, the MCM4 subunit seems to be the closest homologue to the single MCM present in Archaebacteria, which possesses the DNA helicase activity (Kearsey and Labib, 1998; Kelman et al., 1999; Shechter et al., 2000; Chong et al., 2000). Together these data suggest that an MCM4-containing complex can carry out some limited DNA unwinding at DNA replication origins but that the full MCM2-7 complex is required for processive DNA synthesis, as shown in vivo (Labib et al., 2000). We propose a two-step assembly in which RPA contributes to the formation of an open origin complex, while a further processive unwinding by the full helicase complex permits entry of additional RPA molecules and DNA synthesis to proceed. A similar mechanism takes place in prokaryotes, where priming of short nascent strands cannot occur without some helicase action (Fang et al., 1999). In E. coli, the singlestrand DNA-binding protein (SSB) contributes to the opening of OriC but not to the DNA unwinding at this early step.
We have also defined here two discrete stages of DNA replication that are characterized by the synthesis of discrete sizes of nascent DNAs in which chromatin-bound RPA34 is associated. The first stage is defined when DNA replication is blocked at the initiation stage by aphidicolin. Nascent DNAs of 39 nt are synthesized. The second stage is revealed by the depletion of MCM3, in which initiation of DNA replication can proceed up to 320 nt, after which the full MCM helicase complex is required. A discrete nascent DNA population of 120-160 nt is synthesized and contains RNA-primers, as observed at the human lamin B2 origin (Abdurashidova et al., 2000), whereas the second population of 200-320 nt may represent the first two nascent DNA processed and linked, as
RPA is a component of Pre-RCs RNA primers are removed in this process. The accumulation of these two forms may signal abortive DNA synthesis because of the lack of fully processive MCM helicase and possibly specific features of chromatin at origins (Anderson and DePamphilis, 1979; Lipford and Bell, 2001). We are grateful to Y. Adachi for generous gift of polyclonal antibodies and purified RPA complex. We thank M. Peter for providing us with non-degradable B cyclin, N. Lautredou (Cellular Imagery Regional Center) for her assistance with confocal microscopy analysis, and P. Travo, head of the IFR4 Integrated Imaging facility, for his constant interest and support. We also thank D. Fisher, C. Jaulin and P. Pasero for critical reading of this manuscript. This work has been supported by the Association pour la Recherche sur le Cancer (ARC), the Ligue Nationale contre le Cancer, the Foundation pour la Recherche Médicale (FRM), and Human Frontier Science Program.
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