Fig. 7. Requirement of chromatin binding of Cut5 for DNA replication. (A) Sperm nuclei were pre-incubated in S-phase egg extract in the presence of 50 µg/ml p21 and 100 µM roscovitine (p21/R) for 15 min at 23°C. The chromatin fractions were then isolated and incubated in mock- (Cont.+p21/R→mock) and Cut5-depleted (Cont.+p21/R→ ΔCut5) egg extracts at 23°C. At the times indicated, which include 15 min pre-incubation, samples were treated with NP-40, fixed with formaldehyde, and the amount of Cut5 bound to chromatin and the incorporation of Cy3-dCTP were analyzed as described in the legend for Figure 6. As a negative control, sperm nuclei were incubated in the Cut5-depleted egg extract throughout the time-course (ΔCut5). (B) S-CDK-dependent binding of Cut5 to chromatin in the absence of Cdc45. Sperm nuclei were incubated in mock- and Cdc45-depleted egg extracts with or without p21/roscovitine at 23°C. After incubation for the times indicated, samples were analyzed as in (A).