A Role for Ran-GTP and Crm1 in Blocking Re-Replication Ryuji Yamaguchi, John Newport  Cell  Volume 113, Issue 1, Pages 115-125 (April 2003) DOI: 10.1016/S0092-8674(03)00200-9

Figure 1 RanT24N Restores MCM Loading in NPE and Fully Formed Nuclei (A) Analysis of chromatin bound proteins in egg extracts. Sperm chromatin was incubated in egg cytosol (lane 1) or NPE (lanes 2–5) with 20 μM of RanT24N (lane 4) or wild-type Ran (lane 5) for 30 min. No chromatin was added to the sample in lane 2. Chromatin bound proteins were isolated and analyzed by Western blotting. (B) RasN17 did not restore MCM loading in NPE. Sperm chromatin was incubated in NPE with 20 μM of the indicated proteins. Lane 1, untreated; lane 2, RanT24N; lane 3, RasN17. (C) Treatment of NPE with RanGAP and RanBP1 restored MCM loading. Sperm chromatin was incubated either in egg cytosol (lane 1), NPE (lane 2), or NPE pretreated with 50 ng/μl of RanGAP and RanBP1 for 30 and 90 min (lanes 3 and 4, respectively). No DNA was added to NPE in the last sample (lane 5). Chromatin bound proteins were analyzed by Western blotting. We note that with 30 min preincubations, a small amount of MCM was observed associating with chromatin (data not shown). (D) Sperm chromatin was incubated with purified membranes either with cytosol depleted of MCM7 (lanes 2–6) or depleted cytosol mixed with 1/2 volume of complete cytosol (lane 1) for 2 hr. In samples five and six, 50 μg/ml of GST-Kip were added. During the first 2 hr, samples were removed and examined. Nuclear growth rates did not vary greatly from sample to sample. After 2 hr when large intact nuclei had formed, 1/2 volume of complete cytosol was added to samples three, four, and six. Samples two and five were processed for chromatin isolation. After a further 30 min incubation, 20 μM of RanT24N was added to sample four, and incubation continued for another 30 min At 3 hr, samples one, three, four, and six were processed. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 2 RanT24N Induced Re-Replication of Sperm Chromatin (A) Time course of DNA synthesis. 30,000 sperm chromatin was incubated in 10 μl of cytosol for 30 min, followed by addition of 25 μl of NPE supplemented with either 20 μM of RanT24N or an equal volume of PBS. Reactions were stopped at 30, 60, 90, and 120 min, and amounts of DNA synthesized were measured. (B) The samples were prepared as in (A), in the presence of 400 μM BrdUTP, and after 120 min the DNA products were analyzed by CsCl gradients. Fraction 1 is at the top of the tube and fraction 19 at the bottom. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 3 RanT24N Induces Re-Initiation in G2 Nuclei Chromatin bound proteins in reconstituted nuclei. Sperm chromatin was incubated in low-speed egg extracts for the indicated time. At 110 min, 2 μM of RanT24N was added to the extract in one sample. (A) Samples were made with α32PdATP and 400 μM BrdUTP and processed for DNA replication assays. Inserts: analysis of RanT24N-treated sample by CsCl gradient fractionation. HH and HL indicate positions of Heavy-Heavy and Heavy-Light chains, respectively. (B) Nuclear proteins (top) and chromatin bound proteins (bottom) were isolated at the indicated times and analyzed by Western blotting using anti-MCM7 antibody. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 4 RanQ69L Inhibits MCM Loading (A) High concentrations of Ran-GTP are inhibitory for MCM loading. Sperm chromatin was incubated for 30 min in egg cytosol containing 0–20 μM Ran-GTP (lanes 1–3) or 20 μM RanQ69L loaded with GTP (lane 4). In a separate experiment, 20 μM of either GTP or H-RasQ61L loaded with GTP were added to the samples six and seven, respectively. Along with the untreated sample (lane 5) and a sample containing no DNA (lane 8), chromatin was isolated and chromatin-associated proteins analyzed by Western blotting. (B) RanQ69L inhibited DNA replication while RanT24N induced re-replication. Sperm chromatin was incubated in cytosol for 30 min either with 20 μM RanQ69L-GTP (sample two), 20 μM of RanT24N (sample three), or without addition (sample one). Following this, 2.5 volumes of NPE was added and the reaction continued for 2 more hours before DNA synthesis was assayed. (C) Left: preincubation with Kip blocks the inhibitory effect of RanQ69L. Cytosol was preincubated without (lanes 1 and 2) or with (lanes 3 and 4), 50 μg/ml of GST-Kip for 30 min. Sperm chromatin was added to all samples and RanQ69L-GTP to samples in lanes 2 and 3 and incubated for another 30 min. Chromatin-associated proteins were isolated and analyzed by Western blots. Right: once bound to chromatin, MCM is stable. Sperm chromatin incubated in cytosol for 30 min was isolated and reincubated in cytosol lacking MCM7 for 30 min In sample six, 20 μM of RanQ69L-GTP was added. In sample 7, 50 μg/ml of GST-Kip was added. Chromatin bound proteins were analyzed by Western blotting. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 5 Crm1 and Ran-Q69L Form a Complex with MCM (A) RanQ69L and Crm1 coprecipitate with MCM7. 5 μg of the indicated antibody conjugated to protein A-sepharose was mixed with 20 μl of cytosol. Samples three and four also contain 20 μM of RanQ69L-GTP. After 30 min of gentle agitation, beads were washed four times in PBS, resuspended in 100 μl of SDS buffer, and 20 μl were analyzed by Western blotting. (B) Anti-MCM7 antibody precipitation was performed as above in the presence of radiolabeled Crm1 added to NPE. Sample one contained buffer. Sample two, 20 μM RanT24N. Sample three, 20 μM of RanBP1 and RanGAP plus 50μg/ml of GST-Kip for 30 min. Sample four is an immunoprecipitated from a nonspecific rabbit IgG. (C) Top: Western blotting of Xenopus egg cytosol (lane 1) and Crm1-depleted cytosol (lane 2), probed with anti-Crm1 antibody. Bottom: sperm chromatin was incubated in Crm1-depleted cytosol (lane 1), Crm1-depleted cytosol with 20 μM RanQ69L (lane 2), and Crm1-depleted cytosol supplemented with recombinant Crm1 protein at endogenous levels (lane 3). No sperm chromatin was added to sample four. After 30 min, sperm chromatin was isolated, and samples were analyzed by Western blotting. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 6 The Geminin-Cdt1 Pathway Is Independent of the Ran-GTP Pathway (A) Geminin is present at very low levels in egg cytosol: Western blotting of 1 μl of cytosol (lane 1) and 1 μl of NPE (lane 2) by anti-geminin antibody. (B) Geminin inhibition is reversed by Cdt1, but not by Kip, while RanQ69L inhibition is reversed by Kip, but not by Cdt1. Sperm chromatin was incubated with either 80 nM geminin (lanes 2–4) or 20 μM RanQ69L-GTP (lanes 5–7) for 30 min. Samples three and six also contain 200 nM Cdt1. Samples four and seven were preincubated with 50 μg/ml of GST-Kip. Chromatin from all samples was isolated and analyzed by Western blotting. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)

Figure 7 A Model for Ran-GTP-Induced MCM Inhibition In S phase nuclei, cyclin-kinase activity phosphorylates MCM, and this modification causes it to associate with Ran-GTP and Crm1. The formation of this complex sequesters MCM and prevents it from binding to chromatin, thereby inhibiting re-replication. Cell 2003 113, 115-125DOI: (10.1016/S0092-8674(03)00200-9)