DNA-PKcs and ATM Epistatically Suppress DNA End Resection and Hyperactivation of ATR-dependent G-checkpoint in S-phase Irradiated Cells

We previously reported that cells exposed to low doses of ionizing radiation (IR) in the G-phase of the cell cycle activate a checkpoint that is epistatically regulated by ATM and ATR operating as an integrated module. In this module, ATR interphases exclusively with the cell cycle to implement the checkpoint, mainly using CHK1. The ATM/ATR module similarly regulates DNA end-resection at low IR-doses. Strikingly, at high IR-doses, the ATM/ATR coupling relaxes and each kinase exerts independent contributions to resection and the G-checkpoint. DNA-PKcs links to the ATM/ATR module and defects cause hyper-resection and hyperactivation of G-checkpoint at all doses examined. Surprisingly, our present report reveals that cells irradiated in S-phase utilize a different form of wiring between DNA-PKcs/ATM/ATR: The checkpoint activated in G-phase is regulated exclusively by ATR/CHK1; similarly at high and low IR-doses. DNA end-resection supports ATR-activation, but inhibition of ATR leaves resection unchanged. DNA-PKcs and ATM link now epistatically to resection and their inhibition causes hyper-resection and ATR-dependent G-checkpoint hyperactivation at all IR-doses. We propose that DNA-PKcs, ATM and ATR form a modular unit to regulate DSB processing with their crosstalk distinctly organized in S- and G- phase, with strong dependence on DSB load only in G-phase.