Figure 2.

Degradation of Cdt1 results in the loss of licensing activity. (A) Metaphase-arrested extracts were supplemented with CaCl₂ plus or minus MG132 and optionally preincubated for 2 h at 23°C. Sperm nuclei were then added (10 ng DNA/μl) plus or minus recombinant Cdt1 and extracts were incubated for a further 20 min. An aliquot was then transferred to fresh geminin-treated extract and assayed for its ability to support DNA synthesis (upper chart). Chromatin was isolated from the remaining sample and immunoblotted for Mcm7, Cdt1, Orc1 and Cdc6 (lower panel). (B, C) Metaphase-arrested extracts were supplemented with GST-wtCdt1 or GST-NΔ243 constructs, released into interphase with CaCl₂ and incubated for 2 h. (B) At the indicated times, aliquots of whole extract were subjected to SDS–PAGE and immunoblotted for GST (4 ng/μl GST-Cdt1 constructs). (C) After 2 h, sperm nuclei were added (10 ng DNA/μl) and after additional 30 min incubation, chromatin was isolated and blotted for Mcm7 (0.3 ng/μl GST-Cdt1 construct). The left panel shows 3 ng of each construct blotted for GST. (D) Metaphase-arrested extracts were supplemented with sperm nuclei (10 ng DNA/μl) plus or minus MG132 and then released into interphase with CaCl₂. At the indicated times, aliquots of whole extract (upper panels) or isolated chromatin (lower panels) were subjected to SDS–PAGE and immunoblotted for Cdt1, geminin or Mcm7.