ABSTRACT S N 1-alkylating agents, such as the mutagenic and cytotoxic drug N-methyl- NЈ-nitro-N-nitrosoguanidine (MNNG), robustly activate the DNA damage-responsive G 2 checkpoint. Establishment of this checkpoint is dependent on a functional mismatch repair (MMR) system; however, exposure to high doses of MNNG overrides the requirement for MMR to trigger G 2 arrest. In addition, unlike moderate-dose exposure, in which the G 2 checkpoint is attenuated in ataxia-telangiectasia, mutated (ATM)-deficient cells, high-dose MNNG treatment activates G 2 arrest through an ATM-independent mechanism. We document that this arrest is sensitive to the pharmacological agents caffeine and 7-hydroxystaurosporine (UCN-01) that inhibit the checkpoint kinases ATM/ATM and Rad-3-related (ATR) and Chk1/Chk2, respectively. Furthermore, these agents block inactivation of the cell-cycle regulatory molecules Cdc25C and Cdc2, establishing the downstream mechanism through which high-dose MNNG establishes G 2 arrest. Activation of both Chk2 and Chk1 was independent of ATM and MMR in response to high-dose MNNG, unlike the response to moderate doses of this drug. Chk2 was found to be dispensable for cell-cycle arrest in response to high-dose MNNG treatment; however, ATR deficiency and decreased Chk1 expression forced by RNA interference resulted in diminished checkpoint response. These results indicate that MNNG activates the G 2 checkpoint through different mechanisms activated in a dosedependent fashion
