Abstract
Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 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 G1-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.
Highlights
Mimosine is a cell synchronization reagent used for arresting cells in late G1 and S phases
The levels of Cdc45 loading and MCM2 phosphorylation on chromatin increased 1 h after release from Thy 3 Mimo treatment (Fig. 3D). These results suggest that mimosine treatment prevents the transition of the pre-RC to the preinitiation complex by blocking the activation of the pre-RC, which results in synchronization of cells at the G1/S phase boundary
We found that the binding of Proliferating cell nuclear antigen (PCNA) to chromatin was increased in ataxia telangiectasia mutated (ATM)-kd-expressing cells upon mimosine treatment, suggesting that ATM-kd functions as a dominant negative mutation
Summary
Mimosine is a cell synchronization reagent used for arresting cells in late G1 and S phases. Results: Replication fork assembly is reversibly blocked by ATM activation through mimosine-generated reactive oxygen species. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. 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. We show that ATM-mediated cell cycle checkpoint signaling blocks the activation of the pre-RC upon mimosine treatment. We show that the activation of ATM upon mimosine treatment is induced in response to ROSmediated hypoxic stress without DNA damage RNR, ribonucleotide reductase; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; RPA, replication protein A; Thy, thymidine; Mimo, mimosine; kd, kinase-dead; ROS, reactive oxygen species; NAC, N-acetylcysteine. Mimosine Blocks S Phase Entry through ATM Activation suggest that mimosine treatment blocks S phase entry through ATM activation
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