Figure 2.

Cdc6 inhibits DNA replication after origin licensing at an earlier stage of DNA replication. (A and B) GST–Cdc6 (700 nM) was added to the reaction mixture at indicated times after the commencement of incubation of sperm nuclei (3 ng DNA/µl) with egg extracts. Synthesized DNA was measured after 90 min from the start of incubation. (C) A schematic representation of the experimental procedures for D and E. (D) Chromatin was isolated after incubation of sperm nuclei (3 ng DNA/µl) for 20 min with extracts containing buffer (none) or GST–Cdc6 (700 nM) in the presence or absence of geminin (100 nM). Isolated chromatin was subjected to immunoblotting. Open and closed arrowheads represent endogenous Cdc6 and GST–Cdc6, respectively. Egg extracts (Ex; 2 µl) were also applied. (E) Sperm nuclei (15 ng DNA/µl) were incubated with extracts supplemented with buffer (none) or GST–Cdc6 (700 nM) for 20 min in the presence or absence of geminin (100 nM). Chromatin was isolated and incubated for 90 min with fresh geminin-supplemented extracts containing [α-³²P]dATP. DNA synthesis in the second incubation was measured and represented as a percentage of the radioactivity incorporated into DNA in the samples of interest to that after incubation with control extracts for 90 min.