Figure 6

Depletion of Cdc45 or Pol1 by the off -AID system causes tight cell cycle arrest in early S phase. (A) HM3390 cdc25-22 P _nmt81 -cdc45-aid double-TIR1 or HM3397 cdc25-22 P _nmt81 -pol1-aid double-TIR1 strain grown at 25°C was cultured in the presence of thiamine (10 μg/ml) for 4 h or 6 h, respectively, arrested at G2/M phase by incubation at 36°C for 3.5 h, and then released at 25°C (time 0). NAA (0.5 mM) was added 1 h before release. HU (12 mM) was added to non-depleted cells at time 0 (not shown in the figure). (B) Cdc45-aid and Pol1-aid proteins in whole-cell extracts prepared at the indicated time points were analyzed by immunoblotting using anti-Cdc45 and anti-IAA17 (Pol1) antibodies, respectively. Non-specific band is indicated by an asterisk (*). (C) DNA contents of the cells with (right) or without (left) protein depletion were analyzed by flow cytometry.