Radiation-dose-dependent functional synergisms between ATM, ATR and DNA-PKcs in checkpoint control and resection in G2-phase

Abstract

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.

SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase

“…We investigated contributions of SCF SKP2 to resection at DSBs induced by ionizing radiation (IR) by depleting SKP2. Our recent work shows profound differences in the regulation of resection between cells irradiated in S-versus G 2 -phase, as well as at low versus high IR-doses 43,44 . Therefore, we conducted cell-cycle-and IR-dose-dependent analysis.…”

“…At low IR-doses, cell cycle-dependent analysis is possible by IF. S-phase cells are labelled with EdU 44 and resection measured by quantitating chromatin-bound RPA in EdU-negative (EdU − ) cells, in the G 2 -phase compartment ( Fig. S1A).…”

“…S1C). Therefore, we focus here on G 2 -phase cells, but include cells irradiated in G 2 -phase 44 , as well as cells irradiated in S-phase that enter G 2 -phase during the postirradiation incubation period 43 .…”

What p53 sees: ATM and ATR activation through crosstalk between DNA damage response pathways

“…The results shown are more consistent with XP-A and XP-C cells; to model XP-E cells, which are defective in GG-NER but proficient in TC-NER, we would set only k DN = 0. Mladenov et al 2019-1 [133]: This recent work focuses on how the activity of three PI3Ks-ATM, ATR, and DNA-PKcs-changes at high and low doses of γ radiation. At low doses of γ radiation, ATM and ATR cooperate to activate the G2 checkpoint, but at high doses of γ radiation, the cell can activate the G2 checkpoint when treated with either ATRi or ATMi.…”

“…It even violated observations unrelated to crosstalk: ATM did not pass the detectability threshold in response to 10 Gy γ radiation. Despite the kinases responding weakly, [113] Inhibiting ATM slows complex DSB repair [19] ATM responds downstream of ATR to UV damage [31] ATM can robustly respond to as little as 0.4 Gy within 15 minutes [31] Active ATM is not detectable 1 hour after 10 J/m 2 UV exposure, but is after 5 hours [18] Both ATM and ATR localize to DSBs within 10 minutes [124] ATR can localize to DSBs in ATM-deficient cells [117] ATR is activated before DSBs undergo extensive resection [117] Robust post-γ-radiation ATR response requires end resection [20] ATR can be activated by γ radiation in G1 phase [20] ATR focus formation around DSBs depends on ATR kinase activity [131] ATM-or Mre11-deficient cells form fewer ATR foci after γ radiation [133] Inhibiting ATM affects end resection rates during G2 phase Table 9: Changes in the qualitative model validation with or without key processes. Some rows are missing because all eight models satisfied the claims they represent.…”