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Figure 2. High concentrations of cdk2–cyclin E inhibits both DNA replication and the binding of MCM3 to chromatin. (A) Purified egg cytosol containing 1 μM of added cdk2–cyclin E was preincubated for 30 min. After this incubation, membrane and sperm (1,000/μl) were added to the extract. As shown, by using phase optics (left), these sperm formed intact nuclei, and by fluorescent staining (right), the DNA decondensed. (B) Interphase cytosol was preincubated alone (− cdk2–cyclin E), or with 1 μM of cdk2– cyclin E (cdk2–Cyclin E) for 30 min. After this membrane and 1,000 sperm/μl were added. The autoradiogram shows 32P incorporated into DNA during 15 min pulses starting at indicated times after sperm addition. In the absence of cyclin E, replication occurred normally, while in the presence of cyclin E it was strongly inhibited at all time points. (C) cdk2–cyclin E was added to interphase cytosol to the final concentrations indicated. After a 30-min incubation, 1,000 sperm/μl were added to each reaction, the reactions were incubated for another 30 min, and then the sperm were pelleted. The pellets were resuspended in SDS-PAGE sample buffer. MCM3 bound to the sperm chromatin was determined by Western blotting using anti-MCM3 antibody. As shown, MCM3 binding to chromatin was inhibited as the concentration of cdk2–cyclin E increased from 0.12 to 1.0 μM. (D) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. 1 μM of cdk2–cyclin E was then added together with membrane. Photographs show a typical nucleus that formed under these conditions. (E) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. After this, membrane was added with or without 1 μM of cdk2–cyclin E and DNA replication was assayed as in B. Replication in both extracts was identical, demonstrating that late addition of cdk2–cyclin E does not inhibit chromatin from replicating. (F) Sperm was added to cytosol to a final concentration of 1,000 sperm/μl. After a 30-min incubation, the indicated amounts of cdk2–cyclin E were added, and the reaction was incubated for another 30 min. At the end of the incubation the sperm were pelleted and assayed for MCM3 by Western blotting as described in C. As shown, late addition of cdk2–cyclin E does not inhibit binding of MCM3 to chromatin. Bars: (A and D) 10 μm. Image published in: Hua XH et al. (1997) Image reproduced on Xenbase with permission of the publisher and the copyright holder. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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