ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage

Armin M Gamper,Jan H Beumer,Joel S Greenberger,Christopher J Bakkenist,Reza Rofougaran,Simon C Watkins

doi:10.1093/nar/gkt833

Armin M Gamper, Jan H Beumer + Show 4 more

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Journal: Nucleic Acids Research	Publication Date: Sep 14, 2013
Citations: 67	License type: CC BY 3.0

Affiliation: University of Pittsburgh

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The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.

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Ataxia telangiectasia mutated (ATM) and the related kinase ATM- and Rad3-related (ATR) are principal signal transducers that mediate DNA damage signalling
ATR kinase inhibition by ETP-46464 is rapidly reversible as removal of ETP-46464 just 30 min prior to ultraviolet irradiation restores the ability of ATR to phosphorylate Chk1 (Figure 1C)
To see whether the observed replication protein A (RPA) foci in G1 could be at double strand breaks (DSBs), we investigated the incidence of RPA foci in U2OS cells stably expressing green fluorescent protein (GFP)-53BP1 irradiated in G1 phase

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Ataxia telangiectasia mutated (ATM) and the related kinase ATM- and Rad3-related (ATR) are principal signal transducers that mediate DNA damage signalling. While ATM is recruited to DNA double strand breaks (DSBs) by the Mre, Rad and Nbs complex, ATR and its constitutive interacting partner ATRIP bind to replication protein A (RPA)-coated single-stranded DNA (ssDNA). ATR can be further activated by direct interactions with DNA topoisomerase 2-binding protein 1 (TopBP1), which is recruited to ssDNA/ double-stranded DNA junctions by the Rad9-Rad1Hus1 [-1] complex. Claspin-mediated phosphorylation of Chk kinase at serines 317 and 345 by ATR regulates Chk activity [1]. ATR/Chk signalling is initiated at structures containing ssDNA and a junction between ssDNA/double-stranded DNA, and this is associated with S and G2 phase cell cycle checkpoints in mammalian cells [2]

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