Radiation-dose-dependent functional synergisms between ATM, ATR and DNA-PKcs in checkpoint control and resection in G(2)-phase

Using data generated with cells exposed to ionizing-radiation (IR) in G(2)-phase of the cell cycle, we describe dose-dependent interactions between ATM, ATR and DNA-PKcs revealing unknown mechanistic underpinnings for two key facets of the DNA damage response: DSB end-resection and G(2)-checkpoint activation. At low IR-doses that induce low DSB-numbers in the genome, ATM and ATR regulate epistatically the G(2)-checkpoint, with ATR at the output-node, interfacing with the cell-cycle predominantly through Chk1. Strikingly, at low IR-doses, ATM and ATR epistatically regulate also resection, and inhibition of either activity fully suppresses resection. At high IR-doses that induce high DSB-numbers in the genome, the tight ATM/ATR coupling relaxes and independent outputs to G(2)-checkpoint and resection occur. Consequently, both kinases must be inhibited to fully suppress checkpoint activation and resection. DNA-PKcs integrates to the ATM/ATR module by regulating resection at all IR-doses, with defects in DNA-PKcs causing hyper-resection and G(2)-checkpoint hyper-activation. Notably, hyper-resection is absent from other c-NHEJ mutants. Thus, DNA-PKcs specifically regulates resection and adjusts the activation of the ATM/ATR module. We propose that selected DSBs are shepherd by DNA-PKcs from c-NHEJ to resection-dependent pathways for processing under the regulatory supervision of the ATM/ATR module.