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A Hypophosphorylated Form of RPA34 Is a Specific Component of Pre 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, 34396 Montpellier CEDEX 5, France 2Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tûbingen, Germany *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 single- not require nuclear membrane formation, and is sensitive stranded DNA-binding protein required for DNA to the S-CDK inhibitor p21. We also provide evidence that replication. In Xenopus, RPA assembles in nuclear foci that RPA34 is present at initiation complexes formed in the form before DNA synthesis, but their significance in the absence of MCM3, but which contain MCM4. In such assembly of replication initiation complexes has been conditions, replication foci can form, and short RNA- questioned. Here we show that the RPA34 regulatory primed nascent DNAs of discrete size are synthesized. subunit is dephosphorylated at the exit of mitosis and binds These data show that in Xenopus, the hypophosphorylated to chromatin at detergent-resistant replication foci that co- form of RPA34 is a component of the pre-initiation localize with the catalytic RPA70 subunit, at both the complex. initiation and elongation stages of DNA replication. By contrast, the RPA34 phosphorylated form present at Supplementary material available online at mitosis is not chromatin bound. We further demonstrate http://jcs.biologists.org/cgi/content/full/117/21/4909/DC1 that RPA foci assemble on chromatin before initiation of DNA replication at sites functionally defined as initiation Key words: Replication protein A, DNA replication foci, Xenopus, replication sites. Association of RPA with these sites does MCM, Nascent DNA, Aphidicolin Introduction RPA and the DNA polymerase δ processivity factor PCNA Replication protein A (RPA) is a stable complex of three (Adachi and Laemmli, 1992; Dimitrova et al., 1999), but none different subunits (p70, p34 and p11) that participates in of the proteins forming the pre-replication complex (e.g. ORC, different cellular processes: DNA replication, recombination cdc6, cdt1, MCMs) exhibits such clear localization. The and repair (Wold, 1997). The RPA70 subunit has a high affinity significance of RPA foci is therefore debated. for single-stranded DNA, but a DNA-binding activity that is In Xenopus in vitro systems, RPA is present on chromatin associated with the RPA34 and RPA11 subunits (Bochkareva before initiation of DNA replication. It first localizes at distinct et al., 1998). Cell-cycle-regulated phosphorylation of RPA34 foci that might be pre-replication centers, but appears to be re- at the G1-S transition has been described (Din et al., 1990; localized evenly throughout the nucleus after initiation of DNA Fang and Newport, 1993; Pan et al., 1994). However, it is not replication (Adachi and Laemmli, 1992). In mammalian cells, yet clear whether phosphorylation of RPA is involved in the RPA localizes to replication foci at the onset of S phase onset of DNA replication (Pan et al., 1995; Philipova et al., (Brenot-Bosc et al., 1995; Murti et al., 1996), but RPA foci 1996), as DNA replication efficiency is not affected by DNA- were not observed in early G1 (Dimitrova et al., 1999; dependent protein kinase (Brush et al., 1994; Lee and Kim, Dimitrova and Gilbert, 2000). These observations have led to 1995; Pan et al., 1995) or Cdc2 (Henricksen and Wold, 1994), the proposal that RPA foci in Xenopus may be unrelated to two kinases involved in RPA modification. initiation of DNA synthesis and may represent a nonspecific The RPA70 large subunit alone is not sufficient for DNA storage of RPA bound to chromatin (Dimitrova et al., 1999). replication, and the RPA complex cannot be replaced by E. coli We have investigated the association of the regulatory single-stranded DNA-binding protein SSB (Dornreiter et al., subunit RPA34 with chromatin during the cell cycle and its 1992; Walter and Newport, 2000), suggesting that the RPA34 participation in pre-replication complexes. We show that the and RPA11 subunits are necessary for the function of RPA in regulatory RPA34 subunit is rapidly dephosphorylated upon DNA replication. RPA participates in the synthesis and mitosis exit, and then associates with chromatin prior to the processing of Okazaki fragments during DNA replication in initiation of DNA replication. RPA34 is detected in its viruses and in yeast (Mass et al., 1998; Bae et al., 2001). In hypophosphorylated form during the whole of S phase and multicellular organisms, however, its participation in the pre- assembles into detergent-resistant foci. Mitosis results in initiation complex is currently unclear. Notably, sites of DNA phosphorylation of RPA34 and its loss of chromatin binding synthesis are detected as replication foci that co-localize with activity. At mitosis exit, RPA foci first form on sites that are 4910 Journal of Cell Science 117 (21) functionally defined as replication initiation sites. The 37°C. DNA was then extracted with phenol/chloroform and further assembly of these foci do not require nuclear membrane purified by gel filtration chromatography on a spin column (P6, formation. We further show that RPA foci can assemble in BioRad). DNA was then incubated with 2 units of λ-exonuclease MCM3-depleted extracts that still contain MCM4 bound to (Biolabs) for 4 hours at 37°C in a final volume of 40 µl. At the end λ chromatin. Moreover, these RPA foci co-localize with stalled, of the incubation -exonuclease was inactivated by heating at 70°C short, nascent DNAs of discrete size. for 15 minutes. Analysis of the products was done by electrophoresis on a 2% agarose gel. Materials and Methods Indirect immunofluorescence assays Antibodies For analysis of chromatin-bound proteins, 10 or 15 µl samples were The anti RPA34-specific monoclonal antibody (324A.1) was isolated diluted 10 times with 0.3% Triton X-100 in XB and incubated for 5 from a monoclonal antibody library raised against Xenopus oocyte minutes on ice. They were fixed by addition of an equal volume of germinal vesicle proteins (Dreyer et al., 1981) and recognizes only 8% formaldehyde in XB, for 1 hour at 4°C. For analysis of total dephosphorylated RPA34 (Fig. S1, see supplementary material). The nuclear proteins, 10 µl samples were fixed with 200 µl of 4% RPA polyclonal antibodies 309.112 and E-Ky were generous gifts of formaldehyde in XB. Nuclei or chromatin were centrifuged onto glass Y. Adachi (Institute of Cell and Molecular Biology, Edinburgh, UK). coverslips at 1500 g for 8 minutes, through a 0.7 M sucrose cushion. The 309.112 antibody recognizes both RPA70 and RPA34 (Adachi Coverslips were post-fixed for 4 minutes in cold methanol and and Laemmli, 1994) but we observed that it recognized RPA34 only rehydrated for 15 minutes in PBS. After 1 hour saturation at room under its phosphorylated form (Fig. S1, see supplementary material). temperature in PBS, 1% bovine serum albumin (BSA), coverslips The E-Ky antibody is specific for the Xenopus RPA70 subunit were incubated overnight at 4°C with specific antibodies. Each (Adachi and Laemmli, 1992). Antibodies against Cdt1, MCM3 and coverslip was washed 5 times for 20 minutes with 0.1% Tween-20 in MCM4 were previously described (Coué et al., 1996; Coué et al., PBS before incubation with secondary antibodies for 1 hour at room 1998; Maiorano et al., 2000a). γ-H2AX polyclonal antibody was temperature, followed by five 20-minute washes with PBS, 0.1% supplied by Interchim. Tween-20, and a 15 minute incubation in 10 µg/ml Hoechst 33258 in PBS. Xenopus egg extracts Interphase and mitotic (CSF) low-speed (LS) extracts were prepared Microscopy and image analysis according to protocols described in detail previously (Menut et al., Confocal microscopy was performed using a Biorad 1024 CLSM 1999) and available at http://www.igh.cnrs.fr/equip/mechali/. 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 Replication reactions laser sharp 1024 software and processing system. Deconvolution Demembranated sperm nuclei were prepared as described (Menut et imagery was performed on cut sections with a DMR Leica microscope al., 1999). Nuclei were incubated in LS extracts (1000 nuclei/µl), or coupled to a CCD Princeton Camera. 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 Results cycloheximide and reactions were incubated at room temperature, or α32 Two distinct populations of the regulatory RPA34 subunit 18°C for pulse assays. DNA synthesis was measured by [ P]dCTP are present at mitosis and interphase with opposite incorporation as previously described (Menut et al., 1999). DNA replication was also determined by immunofluorescence after 3- chromatin binding activity minute pulses with 20 µM biotin-16-dUTP (Boehringer) at 18°C. Xenopus egg extracts mimic most events occurring during S Immunodepletions were performed as described (Maiorano et al., phase and mitosis and are particularly suitable for biochemical 2000a), except for MCM3 depletions, in which IgGs were coupled to analysis of initiation of DNA replication. Xenopus metaphase- protein-G Sepharose beads at 4°C. arrested egg extracts were supplemented with sperm chromatin and released into interphase by calcium addition. Fig. 1A Purification and analysis of chromatin fractions shows the onset of S phase, whereas RPA modifications and binding to chromatin were analyzed during mitosis exit and S 50 µl samples were diluted with 5 volumes of extract buffer (XB: 100 phase in Fig.
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