Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation.

Fanghua Li,Rositsa Dueva,Martin Stuschke,Beate Timmermann,Emil Mladenov,George Iliakis

doi:10.3390/cells11010063

Fanghua Li, Rositsa Dueva + Show 4 more

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https://doi.org/10.3390/cells11010063

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Abstract

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Highlights

Exposure of cells to ionizing radiation (IR) induces DNA double-strand breaks (DSBs) that cause cell death and cancer [1,2,3,4]
In cells irradiated in the G2 phase, DNA-PKcs integrates with ataxia–telangiectasia mutated (ATM) and ATR in checkpoint regulation but functions as a strong suppressor of checkpoint hyperactivation by enabling checkpoint recovery, both at high and low IR doses [18]
While progression of non-irradiated G1 -phase cells into the cell cycle varied slightly between experiments, the IR-induced delay was always pronounced and clearly measurable. We conclude that this delay is a manifestation of the activation of a checkpoint in the G1 phase, in cells irradiated in the G1 phase

Summary

Introduction

Exposure of cells to ionizing radiation (IR) induces DNA double-strand breaks (DSBs) that cause cell death and cancer [1,2,3,4]. DSBs activate in cells of higher eukaryotes a complex network of signaling pathways known as the DNA damage response (DDR) that senses. DSBs and coordinates their repair with the cellular metabolism, aiming to safeguard genomic stability—or alternatively, to eliminate irreparably damaged cells by inducing apoptosis or senescence [5,6,7]. Two additional members of the PIKK family, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ATM and RAD3 related (ATR), serve distinct functions in c-NHEJ and HR, respectively [12]. While there is ample evidence for the function of ATM, ATR and DNA-PKcs independently of each other in distinct aspects of DDR and DSB repair [12], crosstalk and collaboration are known and presumably serve to optimize

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