Fig. 4.

Hamster chromatin becomes licensed for replication at the end of mitosis. (A-C) Geminin prevents replication of Xenopus sperm (A; 1,000 nuclei/μl) and CHO metaphase chromatin (B; 10,000 nuclei/μl), but has no effect on hamster G1 nuclei (C; 6 hours in G1, 10,000 nuclei/μl). The indicated substrates were incubated in Xenopus egg extracts with (diamonds, 0.2 μg/ml; circles, 2 μg/ml; and triangles, 20 μg/ml) or without (squares) the addition of purified geminin. Aliquots were removed at the indicated times and the amount of replicated DNA was determined by acid precipitation as in Fig. 2. (D-G) Synchronized CHOC 400 cells were permeabilized as in Fig. 2 and incubated in control (D,F) or geminin-supplemented (E,G; 2 μg/ml) Xenopus egg extracts. The percentage of replicated DNA at various time points was determined as in Fig. 2. Similar results were obtained in four independent experiments.