Drosophila ATM and Mre11 Are Essential for the G₂/M Checkpoint Induced by Low-Dose Irradiation

Abstract

Others have suggested recently that the conserved ATM checkpoint kinase is minimally involved in controlling the G₂/M checkpoint in Drosophila that serves to prevent mitotic entry in the presence of DNA damage. Our data indicate that both ATM and its regulator Mre11 are important for the checkpoint and that their roles become essential when animals are challenged with a low dose of X rays or when they have compromised checkpoint function of the ATM-related ATR kinase.

THE cell's response to genotoxic insults involves complex and often redundant signaling pathways. When challenged with excessive DNA damage, multiple cascades could be activated. This could confound interpretation of the precise roles that different factors play in the pathway. For example, the roles of mammalian NBS and H2AX in the G₂/M checkpoint were revealed only when a lower dose of radiation was applied (Buscemi et al. 2001; Fernandez-Capetillo et al. 2002). Recently, three groups concluded that Drosophila ATM is not required for the G₂/M checkpoint in the larval wing (Oikemus et al. 2004; Silva et al. 2004; Song et al. 2004), which would suggest that Drosophila ATM have lost this important checkpoint function through evolution since both mammalian ATM and Mre11 regulate the same checkpoint (Xu et al. 2002; Theunissen et al. 2003). In the earlier Drosophila studies, a dose of 4000 rad of X rays was used to induce the checkpoint. However, a lower dose of 500 rad was sufficient for checkpoint activation (Hari et al. 1995; Laurençon et al. 2003). We set out to investigate the possibility of a dosage effect on the G₂/M checkpoint.

We generated mutations in tefu and mre11 genes, which encode the Drosophila ATM and Mre11 homologs, respectively (Bi et al. 2004). In wild-type imaginal discs, X-ray irradiation induces a steep decline in the number of mitotic cells (Figure 1), indicative of a functional checkpoint. Since the normal length of the G₂ phase has been estimated to be 4.2 hr in the wing discs of third instar larvae (Neufeld et al. 1998), we assayed exclusively the ability of G₂ cells to stop mitotic entry in the presence of DNA damage. We observed a normal checkpoint response induced by 4000 rad of X rays for both tefu and mre11 mutants, but discovered that neither mutant was able to block mitotic entry when irradiated with 500 rad (Figure 1). Therefore, the G₂/M checkpoint induced by low-dose X rays is indeed dependent on ATM and Mre11. We considered two hypotheses to explain the seemingly normal checkpoint response of the mutants when treated with a high dose of X rays. First, the viability of human ATM-deficient cells was known to be extremely sensitive to irradiation (Jorgensen and Shiloh 1996). It might be that 4000 rad led to the death of the majority of mutant cells in the disc so that few were left to enter mitosis. Alternatively, the high-dose radiation might be able to activate redundant checkpoint pathways and bypass the requirement of ATM and Mre11.

Figure 1.

The roles of ATM, Mre11, p53, and ATR in the G₂/M checkpoint. Third instar larvae were irradiated with the indicated dose of X rays: (▪) 0 rad, 0 hr; () 500 rad, 1 hr; (□) 4000 rad, 1 hr. At 1 or 2 hr after irradiation, mitotic cells were visualized by an antiphospho histone H3 antibody in at least 10 wing discs as described (Laurencon et al. 2003). The number of mitoses was counted for each disc and the averages were plotted with the standard deviation shown. The checkpoint assay yielded similar results for both 1- and 2-hr time points. Only those for the 1-hr time point are shown. The genotypes are given at the top of each graph.

To test the first hypothesis, we took advantage of the fact that a p53 mutation is able to suppress cell death in the wing discs of both tefu^−/− and mre11^−/− animals (our unpublished data). In addition, 4000 rad of irradiation did not result in a marked increase of apoptosis in the discs of the double mutants (data not shown). We reasoned that by rescuing the mutant cells from radiation-induced killing so that they could enter mitosis, we might be able to detect a checkpoint loss in the mutants, even when irradiated with 4000 rad. As shown in Figure 1, both tefu p53 and mre11 p53 double mutants behaved similarly to the tefu and mre11 single mutants, respectively. They responded normally to 4000 rad but failed to arrest in response to 500 rad of radiation. This is inconsistent with the first hypothesis. Nevertheless, we reconfirmed the existence of a checkpoint responding to low-dose X rays that was ATM and Mre11 dependent. In addition, we showed that p53 had no role in the checkpoint at either 500 or 4000 rad, extending previous results (Sogame et al. 2003).

For the second hypothesis, we imagined that the alternative checkpoint could be controlled by Mei-41, the Drosophila ATR homolog, since Mei-41 was required for the checkpoint response to either 500 rad (Laurençon et al. 2003) or 4000 rad (Brodsky et al. 2000) of radiation. A tefu mei-41 double mutant, we reasoned, should show a completely defective checkpoint response at either dose of radiation. We made a tefu^stg mei-41^RT1 double mutant in which the mei-41^RT1 allele was a strong loss-of-function allele in regard to the checkpoint function (Brodsky et al. 2000). We also combined tefu^stg with the weaker mei-41^D9 allele (Laurençon et al. 2003). We subjected both double-mutant combinations to the checkpoint assay. As reported previously, mei-41^D9 animals showed an intermediate checkpoint loss at 500 rad (Laurençon et al. 2003). We found that the response to 4000 rad was largely normal in the same animals. More interestingly, both tefu mei-41 double mutants showed complete loss of the checkpoint response at both radiation doses. This supports our earlier suggestion that the requirement of ATM or Mre11 could be bypassed by a higher dose of radiation.

Consistent with earlier results, we observed that the loss of ATR resulted in a complete loss of the checkpoint regardless of radiation dosage or the state of ATM function, which suggests that ATR is the main checkpoint executor and that the checkpoint controlling function of ATM may be executed through ATR. In the presence of small amounts of damage, the checkpoint-activating signal may have to be amplified by ATM and Mre11 to activate ATR. This would explain the requirement of ATM and Mre11 for checkpoint engagement with low-dose X rays. In the presence of large amounts of damage, the signal is sufficient to activate ATR directly. We further propose that due to a partial loss of ATR function in mei-41^D9 cells, checkpoint activation becomes ATM dependent even when damage is highly abundant.

To account for the radiation-dosage-specific cellular responses, we consider two possible mechanisms. First, the abundance of DNA double-strand breaks (DSBs) could be responsible. ATM is activated mainly by DBSs (reviewed in Abraham 2001). We imagine that ATM and Mre11, but not ATR, are highly sensitive to the presence of a small number of DSBs. When cells are irradiated with 500 rad, both ATM and Mre11 are necessary for amplifying the DSB signal to activate ATR. When DSBs become abundant due to high-dose X rays (4000 rad), ATR can then be directly activated. We do not favor this hypothesis as the main mechanism for the dosage-specific response, given the lack of a checkpoint response in 500-rad-irradiated mre11^−/− cells. During the course of our checkpoint analyses, we noted that mre11^−/− cells were highly defective in repairing chromosome breaks induced by X rays. One hour after being irradiated with a dose of 210 rad of X rays, an average mre11^−/− metaphase nucleus from third instar neuroblasts had 3.15 breaks (n = 65 nuclei), significantly more than an average tefu^stg/stg cell (0.98 breaks, n = 82 nuclei, P < 0.0001) or a wild-type cell (0.81 breaks, n = 52, P < 0.0001). In addition, an mre11^−/− nucleus irradiated with 500 rad displayed fragmented mitotic chromosomes beyond recognition (data not shown). Yet this abundance of DSBs was not sufficient to activate ATR to engage the G₂/M checkpoint, which suggests that ATR cannot be activated by DSBs in the absence of Mre11.

In our second hypothesis, we propose that ATR and ATM sense different types of damage. It recently has been shown that ATR activation requires single-stranded DNA (ssDNA) (Costanzo et al. 2003; Zou and Elledge 2003). We imagine that low-dose X rays induce mainly DSBs, thus necessitating ATM and Mre11 for a proper checkpoint response. Perhaps ATM and Mre11 are required for single-strand processing of these DSBs. With a higher dose, extensive ssDNA may be generated either as a direct consequence of irradiation or as a result of aberrant DSB processing that did not require ATM or Mre11. Our model is consistent with a recent finding in yeast suggesting that ATR activation by DNA damage or replication block requires Mre11 for generating long ssDNA tracts (Nakada et al. 2004).