Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Background: Mimosine is a cell synchronization reagent used for arresting cells in late G(1) and S phases. Results: Replication fork assembly is reversibly blocked by ATM activation through mimosine-generated reactive oxygen species. Conclusion: Mimosine induces cell cycle arrest strictly at the G(1)-S phase boundary, which prevents replication fork stalling-induced DNA damage. Significance: These findings provide a novel mechanism of the mimosine-induced G(1) checkpoint.Mimosine is an effective cell synchronization reagent used for arresting cells in late G(1) phase. However, the mechanism underlying mimosine-induced G(1) cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G(1)-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.