Figure 4.

PP2A is required for initiation but not for elongation of DNA replication. (Column 1) PP2A-depleted egg cytosol (EC; 4 μl) containing 2,000 sperm per μl was incubated for 30 min. Then, 12 μl of mock-depleted NPE containing 50 μg/ml aphidicolin (Aph) was added for 20 min. The chromatin was spun through a sucrose cushion (29), washed, and incubated with 10 μl of mock-depleted NPE and [α-³²P]dCTP. Replication was measured after 50 min, when the replication rate was still within the linear range. (Column 2) The incubation was performed as for column 1, except that PP2A-depleted NPE was used for the elongation reaction. (Column 3) The incubation was performed as for column 2, except that PP2A-depleted NPE was also used for the initiation step. The amount of replication observed in columns 1 and 2 was 25% of the replication observed in nuclei (data not shown), which are known to replicate 100% of the input DNA (27). ECd, PP2A-depleted egg cytosol; NPEd, PP2A-depleted NPE; NPEm, mock-depleted NPE.