Figure 3.

PP2A is required for DNA replication in the nucleus-free system but not for binding of ORC or MCM to chromatin. PP2A-depleted egg cytosol (2 μl) supplemented with 4,000 sperm per μl was incubated for 30 min. (A) Then, 15 μl of NPE, which was 90% depleted of PP2A (data not shown) and supplemented with [α-³²P]dCTP, was mixed with the egg cytosol and incubated for 60, 90, 120, and 210 min (open circles). In a parallel experiment, the PP2A-depleted NPE was supplemented with 0.13 μg/μl C subunit (closed circles). In a control experiment, egg cytosol and NPE were mock-depleted with rat IgG, and no C subunit was added (open squares). (B) Sperm chromatin (final concentration of 2,000 sperm per μl) was incubated in PP2A-depleted (lanes 1 and 2) or mock-depleted (lanes 3 and 4) egg cytosol. After 30 min, the chromatin was recovered by centrifugation through a sucrose cushion. NPE supplemented with [α-³²P]dCTP was added, and replication was measured after 60 and 90 min. (B) PP2A-depleted (lanes 5 and 6) or mock-depleted (lanes 7 and 8) egg cytosol was supplemented with purified membranes and [α-³²P]dCTP. Sperm (final concentration of 2,000 sperm per μl) was added, and replication was measured after 60 and 90 min. More (3×) sperm chromatin was loaded in lanes 1–4 than in lanes 5–8. (C) After a 30-min incubation of 4,000 sperm per μl in egg cytosol, sediment from sperm chromatin from 7 μl was collected in a sucrose cushion and analyzed by Western blotting with antibodies against XORC2 (42) and XMCM3 (43). Lane 1, undepleted egg cytosol; lane 2, mock-depleted egg cytosol; lane 3, 6F9-depleted egg cytosol; and lane 4, undepleted egg cytosol lacking sperm.