JCS01361 Supplemental Material

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• Supplemental Figure 1 -

  Fig. S1. (A) Mitotic and interphase Xenopus egg extracts were prepared as described in the Materials and Methods. Xenopus egg extracts (1) or purified Xenopus RPA trimeric complex (Adachi and Laemmli, 1992, lane 2), were analyzed by 10% SDS PAGE followed by immunoblot with the monoclonal antibody, as described in Materials and Methods. Molecular weight markers (kDa) were run in parallel. (B) MALDITOF spectrometry identified 3 peptides by mass fingerprinting corresponding to putative peptides of Xenopus RPA 34 homologue (TC98275 in TIGR database). Peptide sequences are in red and underlined. Tandem Mass Spectrometry confirms the previous MALDITOF results by de novo sequencing of one peptide (highlighted pink). (C) RPA34 isoforms were analyzed by 2D-gel electrophoresis, and detected by immunoblot using either the 309.112 polyclonal antibody (pAb) or the monoclonal antibody (mAb, Materials and Methods). Mitotic extracts were treated with lambda phosphatase (an identical result was obtained after alkaline phosphatase treatment; data not shown).

  Summary The 324A.1 monoclonal antibody recognizes a polypeptide of 34 kDa both in Xenopus egg extracts and a purified fraction of the RPA complex (Fig. S1A) (Adachi and Laemmli, 1994). This protein was identified by MALDI-TOF Mass Spectrometry as the Xenopus homolog of human RPA34 (RFA2, Identification number TC125790 in the TIGR database). To determine the specificity of the monoclonal antibody, Xenopus egg extracts were fractionated by 2D gel electrophoresis (Fig. S1B) and immunoblotted with either the 309.112 polyclonal antibody (pAb) or the monoclonal antibody (mAb). The polyclonal antibody recognizes several RPA34 isoforms only in mitotic egg extracts (pAb, mitotic). These forms disappear after phosphatase treatement (mitotic + phosphatase), indicating that they are phosphorylated isoforms of RPA34. In contrast the monoclonal antibody (mAb) did not cross-react with any proteins in mitotic extracts, while activation of the extracts with calcium resulted in the appearance of a polypeptide which was recognized by the monoclonal antibody (Mitotic + calcium) as two isoforms both resistant to phosphatase treatment (Interphase + phosphatase). Lambda phosphatase treatments as well as alkaline phosphatase treatments gave the same results, whereas the same treatments eliminate cell phosphorylated RPA34 spots present in mitotic extracts (Fig. S1B). These two isoforms might correspond to two RPA34 proteins encoded by two identical RPA34 genes, because the Xenopus laevis genome is pseudodetraploid (Bisbee et al., 1977). A similar case has been previously observed for the C-MYC protein (Lemaitre et al., 1995), although we cannot exclude that these isoforms may also correspond to post-translational modifications other than phosphorylation, that have not yet been described for RPA. From these data, we conclude that the monoclonal antibody is specific for hypophosphorylated RPA34 and that this RPA34 isoform is not recognized by the polyclonal antibody.

  Methods The first dimension was performed using the Multiphor II and IPGphor Pharmacia systems with 13 centimeters pH 4 to 7 strips. Each sample was supplemented with 50 ml lysis buffer (7 M urea, 2 M thiourea, 4% Chaps (w/v), 0.5% Triton X-100 (w/v) 18 mM DTT, 40 mM Tris-HCl) and 200 ml of rehydration buffer (7 M urea, 2 M thiourea, 2% Chaps (w/v), 18 mM DTT). Strips were rehydrated at room temperature overnight or for 12 hours. The first dimension was performed using a multi-step program: 2 hours at 200 volts, 2 hours at 600 volts, and 21 hours at 3500 volts for the Multiphor II, and 1 hour at 500 volts, 1 hour at 1000 volts and 4 hours at 8000 volts for the IPGphor. Strips were then equilibrated by ten minutes washes with each of three different buffers (buffer 1: 50 mM Tris-HCl pH 6.8, 6 M urea, 30% glycerol 2% SDS; buffer 2: 10 mg/ml DTT in buffer 1; buffer 3: 10 mg/ml iodoacetamide pH 8.8 in buffer 1, bromophenol blue) before the second dimension (10% SDS PAGE). For western blots, proteins were transferred onto C extra membrane (Amersham Pharmacia Biotech). ECL reagents (Amersham Pharmacia Biotech) were used for detection of immunoblot signals.

• Supplemental Figure 2 -

  Fig. S2. Detection of DNA repair by g-H2AX staining on nuclei incubated in Xenopus egg extract. Demembranated sperm nuclei were incubated 60 minutes in Xenopus egg extract containing 0.05 unit/ml EcoRI and prepared for immunostaining (see Materials and Methods). RPA staining was detected with RPA 34 monoclonal antibody and repair by g-H2AX staining. DNA was detected by Hoechst fluorescent dye.

  g-H2AX foci were depicted as a good indication of DNA damage in Xenopus egg extracts (Kobayashi et al., 2002). Fig. S2 shows that nuclei exposed to EcoRI are g-H2AX-positive and that RPA accumulates on nuclei treated in these conditions, as previously observed (Kobayashi et al., 2002).

• Supplemental Figure 3 -

  Fig. S3. (A) Experimental scheme. Sperm chromatin was incubated for 60 minutes at 22°C in a low-speed egg extract (LSE) in the presence of aphidicolin (5 mg/ml) to slow down DNA replication and to label replication initiation foci with biotinylated dUTP. After a first wash, biotinylated dUTP was re-added to fresh LSE to control the extension of DNA replication during the elongation phase (row 2). (B) Same as Fig. 4 of the paper except that row 2 represents a control of DNA replication elongation by adding biotinylated dUTP during the elongation phase. Nuclei were analyzed by immunofluorescence at each step of the reaction to detect DNA (DAPI staining), RPA (monoclonal antibody), and dUTP incorporation.