The main function of ATR CHK

The main function of ATR/CHK1 signaling is activating Lonafarnib checkpoint arrest for S and G2 phases in mammalian cells. There are three checkpoints in response to DNA damage: G1/S, G2/M, and S-phase. The G2/M checkpoint can prevent cells that incur DNA damage in G2 phase or progress into G2 phase from entering mitosis with DNA damage [13], [14]. The molecular mechanism behind IR-induced G2/M arrest varies according to which phase the cells are in the cell cycle at the time of irradiation, and/or the time after irradiation. Cells in G2 phase at the time of irradiation can rapidly arrest to avoid division with DNA damage. This early G2 arrest is ATM-dependent with a threshold dose of about 0.2–0.3Gy [15]. A few papers have reported that the ATR pathway may also be involved [16], [17]. A late and dose-dependent arrest in G2 for cells that were in S or G1 phase at the time of irradiation is ATM-independent, while dependent on ATR and CHK1 [18].
Materials and methods
Results
Discussion ATM-dependent initiation of the G2/M checkpoint induced by IR is well established [13], [15], [18]. However, we observed a dose- and LET-dependent increase in early G2/M arrest in ATM deficient/mutant cells following radiation (Figs. 1 and 6A, Fig. S2A–D, and Table 1). Using specific inhibitors or siRNAs, we found that the ATR pathway and DNA-PKcs were both effective in attenuating the early G2/M arrest (Fig. 2A and C, Fig. S2E and F). Previous publications have suggested that ATR and DNA-PKcs can contribute to regulation of early mitotic entry in the absence of ATM [17], [23]. However, we ruled out a role for DNA-PKcs in the early G2/M checkpoint using a mitotic entry assay (Fig. 2B and D, Fig. S2G–J). DNA-PK plays a prominent role in DSB repair through its role in non-homologous end-joining. In addition, it has also been reported to play a role in normal cell cycle progression through mitosis, which is closely related to the spindle apparatus at centrosomes and kinetochores. Depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity can lead to delayed mitotic transition because of chromosome misalignment [37], [38]. These results support our observations that DNA-PKcs acts through interfering with mitotic exit to enhance the percentage of cells in M phase following IR. In this way, we provide evidence that the ATR pathway plays a pivotal role in the regulation of IR-induced early G2/M checkpoint when cells are deficient/mutant in ATM. Furthermore, our study proves that even in normal cells, ATR signaling can be rapidly activated and function following IR as well as in ATM-deficient/mutant cells or cells with inefficiently activated ATM (Figs. 1, 4, 6A and B). The mechanisms underlying the activation of ATR signaling by IR have not been fully elucidated. It has been reported that there is a requirement for ATM, NBS1 (Nijmegen breakage syndrome gene) and MRE11 (meiotic recombination 11) in the generation of RPA-coated ssDNA, which in turn is necessary for ATR recruitment and CHK1 phosphorylation, accounting for the sequential activation of ATM, ATR and CHK1 at very early times in the response to IR or microlaser treatment [27], [28]. Recent work showed that NBS1 can directly interact with ATR and recruit ATR to the damage site independently of MRE11 and TOPBP1 [12], [39]. Liu reported that Nek1 (NIMA (never in mitosis gene a)-related kinase 1) interacts with ATR-ATRIP and primes the ATR signaling pathway for efficient DNA damage signaling [40]. Our studies using different particle radiation show that the complexity of DSBs is a crucial factor enhancing end-resection, thereby efficiently triggering ATR pathway activation and HR repair (Fig. 4) [9]. Since DSB processing is a prerequisite for ATR activation following IR, we also confirmed that nucleases required for resection are critical for the activation of ATR following irradiation regardless of ATM function after high doses (Fig. 3A and B). Recently Shibata et al. have reported that specific MRE11 endonuclease inhibition initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR [4]. However, the mechanism for the activation of ATR following irradiation, which we think may be different in the presence or absence of ATM, and following different types of irradiation (low and high LETs), is complex and has not been well explored. More work is required to shed light on the mechanisms underlying the activation of ATR signaling following different types of radiation.