Cell cycle inertia underlies a bifurcation in cell fates after DNA damage.

The G-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G-S transition, cells triggering the DNA damage checkpoint in G phase route back to quiescence, and this cellular rerouting can be initiated at any point in G phase. Furthermore, we find that most of the cells receiving damage in G phase actually fail to arrest and proceed through the G-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G phase and indicate that cells have a G checkpoint.