Fig. 4.

Cdc6 mutated for ATPase or Cy is unable to promote recovery from S arrest or reactivation of Cdk2 in vivo. (A) p27^−/− MEF cells inducible for Cdc6, Cdc6^WA, Cdc6^WB, or Cdc6^Cy were synchronized to mid-S phase with or without doxycycline, treated with MMS, and monitored for S-phase progression by flow cytometry and pulse BrdU incorporation. Percentage of BrdU incorporation at 0 h was measured without MMS treatment and therefore represents the population of S phase-progressing cells at the start of the experiments. (B) Each cell harvested in A was analyzed for the levels of Cdc6, S345-phosphorylated CHK1, CHK1, Cdk2, and p21 and the activity and amount of Cdk2, coimmunoprecipitated Cdc6 and p21. Cdk2 served as a loading control in this experiment.