Figure 5.

Cdt1 addition in G2 induces functional re-licensing. Sperm nuclei were incubated at 10 ng DNA/μl in metaphase-arrested extracts supplemented with CaCl₂. After 90 min, extracts were optionally supplemented with recombinant Cdt1. (A) After 120 min, chromatin was isolated and immunoblotted for Cdt1, Mcm7, geminin and Cdc6. The concentration of added Cdt1 was 10 ng/μl. (B) As (A), with 0, 1.25, 2.5, 5 and 10 ng/μl Cdt1. (C) After 120 min, nuclei were isolated and transferred to interphase extract containing [α-³²P]dATP and recombinant geminin. After a further 90 min incubation, DNA synthesis was assessed. As controls, naïve sperm nuclei and licensed chromatin (the 20 min sample from (A)) were incubated in the second extract. The concentration of added Cdt1 was 10 ng/μl.