Interplay between ATM and ATR for the regulation of cellular responses to DNA damage

Cell cycle checkpoints are critical regulatory processes ensuring the proper traversal of a cell through the cell cycle. Activation of checkpoints in response to different types of DNA damage arrests the normal cell cycle progression at different stages, presumably to allow time for repair. ATM (Ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are critical players in G2 checkpoint response. It is still unknown whether the contribution of ATM and ATR to checkpoint activation is through independent inputs from parallel signaling cascades, or whether the two kinases contribute in a sequential manner, in the sense that the activation of one is enhanced by the activation of the other. Here, we use flow cytometry-based measurement of DNA-content and mitotic index variations to detect G2 checkpoint activation and maintenance after exposure of cells with different genetic background to ionizing radiation (IR). Extensive experiments using different cell lines and an array of inhibitors confirm that ATR signaling plays a dominant role in the development of a full scale G2 arrest, while ATM signaling ensures the maintenance of a full scale G2 arrest. In response to IR, ATRIP (ATR interacting protein) and ATM phosphorylated at serine 1981 (ATM-pS1981) show local accumulation on chromatin that is detected in the form foci by immunofluorescence microscopy. Immunofluorescence detection of ATRIP and ATM-pS1981 foci reveals a linkage between foci formation and G2 checkpoint activation. Although ATR plays a dominant role in G2 checkpoint signaling, its activation for G2 arrest may not coincide with foci formation since visible foci form relatively late (1 h after IR). The contribution of ATM to G2 arrest, as indicated by the formation of ATM-pS1981 foci, is strongly dependent on the cell cycle phase in which the cell is irradiated. To investigate this aspect in greater detail, we introduced multicolor staining protocols utilizing appropriate cell cycle makers that allow cell cycle stage characterization before and after irradiation. By combining ATM-pS1981 foci kinetics with cell cycle information, we observed an apparent requirement for about 20 foci for the maintenance of the G2 arrest for cells irradiated in the G2-phase. There is no apparent correlation between ATM-pS1981 foci and G2 arrest for cells irradiated in the G1-phase, as in this case foci disappear relatively quickly. Interestingly, cells irradiated throughout S-phase, show a response similar to that observed with G2 cells and not, as one would expect, a mixed response reflecting subpopulations responding like G1 and subpopulations responding like G2 cells.


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