Figure 4.

The intra-S phase checkpoint reduces the number of active replication forks.

(A, B) Sperm nuclei were incubated at 10 ng DNA/μl in interphase extract with or without 7.5 μM or 15 μM aphidicolin, minus (A) or plus (B) 5 mM caffeine. At early S-phase (35min), nuclei were isolated and transferred to fresh extract supplemented with [α-³²P]dATP and 0.5 mM roscovitine. At the indicated times after transfer (1-30min), total DNA synthesis was measured. (C, D) Sperm nuclei were incubated in interphase extract at 10 ng DNA/μl supplemented with 40 μM aphidicolin minus (C) or plus (D) caffeine. Sperm chromatin was incubated for 35, 45 or 60 min, and was then isolated and transferred to fresh extract supplemented with [α-³²P]dATP and 0.5 mM roscovitine. Total DNA synthesis was measured at different times after transfer.