Ataxia Telangiectasia Mutated (ATM) and ATM and Rad3-related Protein Exhibit Selective Target Specificities in Response to Different Forms of DNA Damage

Christopher E Helt,William A Cliby,Peter C Keng,Robert A Bambara,Michael A O'Reilly

doi:10.1074/jbc.m410873200

Abstract

The ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3-related) protein kinases exert cell cycle delay, in part, by phosphorylating Checkpoint kinase (Chk) 1, Chk2, and p53. It is well established that ATR is activated following UV light-induced DNA damage such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones, whereas ATM is activated in response to double strand DNA breaks. Here we clarify the activation of these kinases in cells exposed to IR, UV, and hyperoxia, a condition of chronic oxidative stress resulting in clastogenic DNA damage. Phosphorylation on Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following oxidative damage by IR involved both ATM and ATR. In response to ultraviolet radiation-induced stalled replication forks, phosphorylation on Chk1 and p53 required ATR, whereas Chk2 required ATM. Cells exposed to hyperoxia exhibited growth delay in G1, S, and G2 that was disrupted by wortmannin. Consistent with ATM or ATR activation, hyperoxia induced wortmannin-sensitive phosphorylation of Chk1, Chk2, and p53. By using ATM- and ATR-defective cells, phosphorylation on Chk1, Chk2, and p53 was found to be ATM-dependent, whereas ATR also contributed to Chk1 phosphorylation. These data reveal activated ATM and ATR exhibit selective substrate specificity in response to different genotoxic agents.

Highlights

Cell cycle checkpoints are initiated in an attempt to prevent the fixation of mutations from one cell generation to the
To determine whether phosphoinositide 3-kinase-related kinases (PIKKs) were required for this growth arrest, A549 and H1299 cells were exposed to room air or hyperoxia for 48 h in presence of the PIKK inhibitor wortmannin
DNA strand breaks caused by IR activate the ataxiatelangiectasia mutated (ATM)/Chk2 pathway, whereas UV lesions and blocks in DNA replication activate ATR/Chk1

Summary

Introduction

Cell cycle checkpoints are initiated in an attempt to prevent the fixation of mutations from one cell generation to the next. ATM and ATR are recruited to different complexes following genotoxic stress Activation of these kinases by damaged DNA results in the direct or indirect phosphorylation or activation of a number of downstream checkpoint controls, DNA repair, or apoptosispromoting targets including p53, Chk, Chk, Brca-1, Rad, and Rad17 [7, 11,12,13]. Activation of the Chk, Chk, and p53 checkpoint proteins is necessary for maintaining genome stability and overall cellular viability following genotoxic stress. Whereas both ATM and ATR are able to target all three of these checkpoint proteins, it remains unclear why different genotoxic stresses result in their specific activation and ability to differentially phosphorylate downstream targets. These findings suggest that the ATM and ATR kinases evolved to respond to unique forms of genotoxic stress

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