Abstract
Using data generated with cells exposed to ionizing-radiation (IR) in G2-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 G2-checkpoint activation. At low IR-doses that induce low DSB-numbers in the genome, ATM and ATR regulate epistatically the G2-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 G2-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 G2-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.
Highlights
The network of cellular responses to DNA damage (DDR)[1,2,3] is built around three kinases of the phosphoinositide3-kinase (PI3K)-related family of protein kinases (PIKKs): ataxia-telangiectasia mutated (ATM), ATM and RAD3-related (ATR) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)[4]
We focused our experiments on the activation of the DNA damage checkpoint and the induction of resection at DSBs in cells exposed to IR in the G2–phase of the cell cycle, where all DSB processing pathways are functional
Exposure of actively proliferating hTert immortalized normal human fibroblasts 82–6 (82–6 hTert) to a low IR dose (2 Gy) causes a precipitous drop of the normalized mitotic index (MI) at 1 h that reflects the prompt activation of the G2-checkpoint (Fig. 1A)
Summary
The network of cellular responses to DNA damage (DDR)[1,2,3] is built around three kinases of the phosphoinositide3-kinase (PI3K)-related family of protein kinases (PIKKs): ataxia-telangiectasia mutated (ATM), ATM and RAD3-related (ATR) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)[4]. ATM, ATR, and DNA-PKcs are large polypeptides with similar domain organizations and common structural features, including a C-terminally located kinase domain and N-terminally located HEAT-repeat domains that mediate protein-protein interactions[5]. They all show preference for phosphorylating serine or threonine residues followed by a glutamine (S/T-Q) and share as a result substrates and functions in DDR. Activated DNA-PKcs phosphorylates various substrates including itself and other c-NHEJ factors, and acts by facilitating the formation of short-range synaptic complexes[18] It has a major role in promoting classical non-homologous end-joining (c-NHEJ), considered the main pathway for repairing DSBs throughout the cell cycle[19,20], with a presumed, small contribution being made by homologous recombination repair (HRR) during S and G2-phase[21,22]. The kinase of TIP60/Kat[9] acetyltransferase is c-Abl[37], itself an ATM substrate[39,40]
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