We also show that in both

We also show that in both ATM proficient and deficient/mutant DBeQ sale the activation of ATR signaling is DSB complexity-dependent (Figs. 1, 4A, C and E, 6A and B). Wang et al. have also reported that the effects of ATR and CHK1 on radiosensitivity are independent of the NHEJ repair pathway [41], [42]. By monitoring survival following low and high LET radiation, they concluded that the checkpoint response plays a more protective role in HZE particle-irradiated cells than in X ray-irradiated cells [43], which is supported by our data in Fig. 6C and D that compared with X ray exposure, ATR has a bigger contribution that ATM following carbon ion beams. Researchers have revealed from different angles that there is an ATM to ATR switch with distinct DNA structures at resected DSBs [30], [44], [45], [46], [47], [48]. Shiotani and Zou devised an in vitro ATM/ATR activation assay using human cell extracts and defined DNA structures, which pointed out that ATM and ATR are activated by similar yet distinct DNA structures at resected DSBs. Although both ATM and ATR depend on the junctions of single- and double-stranded DNA for activation, they are oppositely regulated by the lengthening of single-stranded overhangs (SSOs). Progressive resection of DSBs directly promotes an ATM to ATR switch in vitro, which happens temporally, spatially, and quantitatively for the regulation of ATM and ATR, thus orchestrating the collective checkpoint response in human cells [49]. Our data confirm this speculation from the level of ATR activation that complex DNA ends structures and/or clustered DNA damage predominantly produced by heavy ion beams results in massive DSB resection, which finally leads to more efficient activation of ATR signaling. However, further work is needed to explore how ATM and ATR interface in response to the different DNA damage induced by different types of radiation.
Conflict of interest statement
Acknowledgments We thank Dr. Bing Wang, Dr. Yasuharu Ninomiya, Dr. Akira Fujimori, Dr. Masao Suzuki, Dr. Hiroshi Fujisawa, Dr. Ayako Kariya and Dr. Huizi Li for technical help. This work was supported by ‘National Natural Science Foundation of China (No. 81102076)’, ‘National Natural Science Foundation of China (No. 11475125)’, ‘Universities Natural Science Foundation of Jiangsu Province (No. BK20131165)’, ‘Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)’, ‘Scientific Research Foundation for Returned Scholars, Soochow University (No. Q412600711)’ and JSPS KAKENHI no. 24249067.
The ATR kinase was identified as a member of the PI3-kinase family related to both ATM and Rad 3 . These kinases have been extensively reviewed both in this issue and elsewhere . ATM and ATR are both structurally and functionally related with an overlapping list of specific substrates . Of particular relevance to this review is the difference between the stresses that ATM and ATR respond to. ATM is activated by DNA double-strand breaks caused by agents such as ionizing radiation or chemotherapeutic drugs, whilst ATR is activated by stresses that induce a replication-type insult such as hydroxyurea treatment, ultraviolet light and hypoxia. In response to hypoxia, ATR re-localizes to form nuclear foci and has been shown to phosphorylate many of its known downstream target molecules, including Nbs 1, Rad 17, Chk 1, p53 and histone H2AX , . Hypoxia is a broad term used to encompass a range of oxygen tensions that differ from those found in healthy tissues. Regions of hypoxia occur not only in disease states but also during normal development. Mammalian embryos develop for significant periods of time in an almost entirely hypoxic environment before a blood supply becomes established . Within a tumor, the level of oxygen present is determined by the distance of a cell from the nearest blood vessel. As tumor vasculature is inefficiently formed and leaky, hypoxia, to relative degrees, is a common and serious inevitability. Many studies have shown that the less oxygenated a tumor, the worse the prognosis , , . This has been attributed to the need for efficient vasculature to deliver chemotherapeutic drugs and the reliance of radiation therapy on the production of oxygen radicals to produce DNA strand breaks. Hypoxia is also a physiological inducer of the p53 tumor suppressor gene product and can act as a selective pressure during tumor growth for the elimination of cells with wild-type p53 and the clonal expansion of cells with mutant or otherwise inactive p53 protein , . Detailed measurements indicate that the ranges of oxygen concentrations within a tumor are 8% to almost anoxia (0%). This variation in oxygen tension, combined with the numerous cell lines available and the difficulty in making exact oxygen concentration measurements have led to many conflicting reports. Most studies have, however, highlighted the oxygen dependency of the cellular response to hypoxia. In this review we will concentrate on extreme levels of hypoxia (oxygen concentrations of 0.02%). At these low oxygen levels, that are found in human and rodent tumors, cells undergo a rapid and dramatic S-phase or replication arrest. In addition, cells initially seem to arrest in all cell-cycle phases with an accumulation in G1 after extended periods of time . There are many detailed studies describing hypoxia-induced arrest at the G1/S boundary which will not be discussed here except to note that sometimes cells are described as showing an S-phase arrest in response to hypoxia when in actual fact they are only arrested at the G1/S boundary, i.e. they are prevented from entering S-phase . For the purpose of this review we are focused on cells which are actively synthesizing DNA, and then stop synthesizing in response to severe hypoxia/anoxia.