Figure 6.

Cdt1 addition in G2 induces DNA synthesis and checkpoint activation. (A) Sperm nuclei were incubated at 10 ng DNA/μl in metaphase-arrested extracts supplemented with CaCl₂. After 90 min, extracts were supplemented with 10 ng/μl recombinant Cdt1 and/or 5 mM caffeine. After a further 30 min, nuclei were isolated and immunoblotted for phospho-Chk1 (Ser342). As a positive control, 40 μM aphidicolin was added to the extracts at the start of the incubation. (B) Sperm nuclei were incubated at 3 ng DNA/μl in metaphase-arrested extracts supplemented with CaCl₂. After 90 min, extracts were supplemented with [α-³²P]dATP and various concentrations of recombinant Cdt1 (0, 1.25, 2.5, 5 and 10 ng/μl); at 120 min, the extract was optionally supplemented with 5 mM caffeine. At 240 min, additional DNA synthesis was assessed. (C) Sperm nuclei were incubated at 4 ng DNA/μl in metaphase-arrested extracts supplemented with [α-³²P]dATP and CaCl₂. After 90 min, extracts were optionally supplemented with 10 ng/μl recombinant Cdt1 (filled symbols) and, at 120 min, extracts were optionally supplemented with 5 mM caffeine (squares). Open circles show samples with no addition. At the indicated times, DNA synthesis was assessed.