Regular article
Acute methylmercury exposure induces inactivation of ATR pathway and enhancement of DNA replication stress through degradation of Rad17
2023 Volume 3 Issue 2 Pages reg21-reg28
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2023 Volume 3 Issue 2 Pages reg21-reg28
Methylmercury (MeHg) is a neurotoxic chemical of significant public health concern. Acute exposure to MeHg causes inhibition of cellular proliferation and DNA replication in cellular and animal models, and impedes S-phase entry and progression in several types of primary and cultured cells. However, the effect of MeHg on the DNA damage response remains unknown. The ATM and ATR pathways are two major components of the DNA damage response that regulate cell cycle progression. Here we report that acute MeHg exposure enhanced DNA replication stress in S phase. MeHg also inhibited the kinase activity of ATR, suggesting that replication forks collapsed in S phase. Moreover, MeHg promoted the degradation of Rad17 as a possible mechanism underlying the inactivation of ATR. These results suggest a novel connection between acute MeHg exposure and the ATR pathway, providing insight into a yet unidentified mechanism of the inactivation of Rad17 and the ATR pathway.
MeHg, methylmercury; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related; DSB, DNA double-strand break; UV, ultraviolet; 9-1-1 complex, Rad9-Hus1-Rad1 complex
ATM- and ATR-dependent checkpoint signaling pathways are two major components of the DNA damage response. The kinase activity of ATM is activated by DNA double-strand breaks (DSBs), leading to the phosphorylation of downstream substrates, including Chk2 and Kap1, to promote DSB repair. Various chemically modified DNA, including DSBs, are converted into single-stranded DNAs. Single-stranded DNA regions in the genome, which are also generated by the inhibition of DNA replication through polymerase-helicase uncoupling, activate the ATR pathway, and the ATR pathway stabilizes arrested replication forks. Thus, the inhibition of ATR leads to the collapse of replication forks, resulting in the conversion of the arrested forks into DSBs. ATR phosphorylates Chk1 and downstream substrates to promote cell cycle arrest [1-3].
Rad17 initiates the activation of the ATR pathway. It recognizes a junction between single-stranded DNA and double-stranded DNA to bind damaged chromatin and interacts with the Rad9-Hus1-Rad1 complex (9-1-1 complex) to load the complex onto the damaged chromatin [4-6]. The 9-1-1 complex recruits TopBP1, and this interaction activates the kinase activity of ATR [7, 8]. The activated checkpoint proteins are inactivated by dephosphorylation and proteasomal degradation; Rad17 [9], Chk1 [10], and Claspin [11-13] have been reported to undergo proteasome-dependent protein degradation. We recently showed that the degradation of Rad17 is promoted by the nuclear translocation of Rad17 through the N-terminal D-boxes [14].
Methylmercury (MeHg) is a neurotoxic chemical of significant public health concern according to the World Health Organization [15, 16]. Acute MeHg exposure causes inhibition of cellular proliferation and DNA replication in cellular models [17-19], hinders DNA replication in the developing brain of a rat model [19], and impedes S-phase progression in murine erythroleukemia cells [20]. The inhibition of S-phase entry due to acute MeHg exposure has also been observed in primary cerebellar granule and cortical precursor cells, with a reduction in cyclin E protein levels [19]. The p21 protein, an inhibitor of cyclin-dependent kinases, has been suggested to be involved in cell cycle arrest after MeHg exposure [21]. The toxicity of MeHg is at least partly attributed to the generation of reactive oxygen species [22], and oxidative DNA damage is a major type of DNA damage targeted by the ATM and ATR pathways. Yet, little is known about the effects of acute MeHg exposure on the ATM and ATR pathways. Here we demonstrate that acute MeHg exposure enhances DNA replication stress in S phase.
Cell culture and plasmids
COS-1 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose (D5796, Sigma-Aldrich) supplemented with 5% bovine serum (16170-078, Gibco) and 1% fetal bovine serum (FB-1290/500, Biosera). Human Rad17 (isoform 1, NCBI NP_579921.1) was cloned in a pcDNA3 vector with an N-terminal flag tag [6].
Antibodies
The following antibodies were used: FLAG (PM020, Medical & Biological Laboratories), Hsc70 (sc7298, Santa Cruz Biotechnology), Chk1 (2360, Cell Signaling Technology), Chk1-pSer345 (2348, Cell Signaling Technology), Chk2-pThr68 (2661, Cell Signaling Technology), Kap1 (A300-274A, Bethyl Laboratories), and Kap1-pSer824 (A300-767A, Bethyl Laboratories).
Effect of MeHg on the ATM pathway
COS-1 cells were seeded at 2 × 105 cells per well in 12-well plates, incubated overnight at 37 °C with 5% CO2, and treated with 4 mM thymidine for 20 hours. The cells were exposed to 20‒40 μM MeHg (M0589, Tokyo Chemical Industry) or 5 mM caffeine for 5 hours in the presence of thymidine, and SDS-lysate was prepared. Mock and thymidine-exposed samples were prepared in duplicate for each experiment.
Effect of MeHg on Chk1
COS-1 cells were seeded at 4 × 105 cells in 35-mm dishes and incubated overnight. The medium was removed, and the cells were irradiated with 30 J/m2 UV-C (256 nm) at 135 μw/cm2. The UV-irradiated cells were exposed to 10‒40 μM MeHg or 5 mM caffeine for 5 hours, and SDS-lysate was prepared. Samples were prepared in singlicate or duplicate for each experiment.
Effect of MeHg on Rad17
COS-1 cells were seeded at 4 × 105 cells in 35-mm dishes and incubated overnight. Cells were transfected with 1 μg of pcDNA3/flag-Rad17 or pcDNA4-TO vectors with 5 μL Lipofectamine 2000 (11668-019, Invitrogen). Twenty-four hours after transfection, the cells were divided into four wells of a 24-well plate and incubated overnight. The cells were exposed to 10‒40 μM MeHg for 6 hours, and SDS-lysate was prepared. Mock-exposed samples were prepared in duplicate for each experiment.
MTS assay
COS-1 cells were seeded at 2 × 104 cells per well in 96-well plates and cultured overnight. The cells were exposed to 20 or 40 μM MeHg for 5 hours. The medium was changed to fresh medium, and 20 μL of CellTiter 96 solution (G3580, Promega) was added to each well. The cells were incubated for 3 hours, and the absorbance at 490 nm was measured using a SpectraMax ABS (Molecular Devices). Cell viability was calculated and presented as mean ± standard deviation.
Acute exposure to MeHg induced caspase-independent cell death in COS-1 cells
We evaluated the effect of acute exposure to MeHg in COS-1 cells, which are derived from the kidney of an African green monkey. This cell line has been immortalized using SV40 large T antigen and is not derived from tumors. The effects of MeHg on cell viability were examined by the MTS assay. The exposure to 20 and 40 μM MeHg decreased cell viability to 63% and 7%, respectively (Figure 1). Previous studies have shown that MeHg-induced cell death involves both caspase-dependent and caspase-independent pathways [23, 24]. We examined the effect of caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO on MeHg-induced cell death. However, these inhibitors did not rescue cell viability (data not shown), suggesting that MeHg-induced cell death is not caspase-dependent under the present experimental conditions.
COS-1 cells were exposed to 20 or 40 μM MeHg for 5 hours, and cell viability was examined by the MTS assay. The graph represents results from six independent experiments. P values were calculated using Student’s t-test.***, p < 0.001.
(A) COS-1 cells were exposed to thymidine for 20 hours, followed by exposure to MeHg for 5 hours. SDS-lysate was prepared and probed with the indicated antibodies. Caffeine was used as a positive control.
(B, C) Graphs represent results from more than three independent experiments. P values were calculated using a paired t-test. *, p < 0.05. **, p < 0.01. ***, p < 0.001.
MeHg exposure activated the ATM pathway in the presence of replication stress
We evaluated the effect of MeHg on the ATM pathway. ATM phosphorylates Kap1-S824 in response to DSBs [25]. Thymidine exposure induces replication stress through the depletion of dCTP [26]. The thymidine-exposed cells showed a significant increase in Kap1 and Chk2 phosphorylation (Figures 2A‒C), consistent with our previous study [27]. The thymidine-induced increase in Kap1 phosphorylation was further enhanced by caffeine exposure (Figure 2B, lanes b and c). Previous studies have reported that caffeine inhibits ATR and ATM in vitro but acts as an activator of these kinases in vivo [27, 28]. This discrepancy is attributed to the fact that the inhibition of ATR under replication stress results in replication fork collapse to activate ATM (Figure 5). MeHg exposure also induced Kap1 and Chk2 phosphorylation (Figures 2B, C, lanes g and j). Co-exposure to MeHg and thymidine resulted in significantly higher phosphorylation of Kap1 and Chk2 (Figures 2B, C, lanes f and i). In other words, MeHg and thymidine had a synergistic effect on Kap1 and Chk2 phosphorylation (Figure 2B, lanes f and i), suggesting that MeHg exposure enhances the kinase activity of ATM. Our anti-Chk2 antibody did not detect the Chk2 protein in COS-1 cells (data not shown). These results raised the possibility that the arrested replication forks due to thymidine exposure were converted into DSBs through replication fork collapse upon MeHg exposure.
MeHg exposure inactivated the ATR-Chk1 pathway
The synergistic effect of MeHg and thymidine raised the possibility that MeHg exposure might have led to the inactivation of the ATR pathway. We examined the effect of MeHg on the ATR pathway following UV irradiation. Chk1-S345 is a well-known ATR-dependent phosphorylation site [1]. As reported previously, UV irradiation induced Chk1 phosphorylation in COS-1 cells, which was suppressed by caffeine exposure (Figures 3A, B). These results suggest that the kinase activity of ATR was inhibited by caffeine. Moreover, MeHg exposure decreased Chk1 phosphorylation (Figures 3C, D), suggesting that MeHg inhibits ATR-dependent Chk1 phosphorylation and induces replication fork collapse by inhibiting ATR in the presence of replication stress (Figure 2).
MeHg exposure promoted degradation of Rad17
We examined the effect of MeHg on the components of the ATR pathway to elucidate the mechanism by which MeHg inactivates ATR-Chk1 signaling. Chk1 has been reported to undergo proteasome-dependent protein degradation [10]; however, MeHg exposure did not decrease the amount of Chk1 (Figure 3C). Rad17 is essential for activating and maintaining the ATR-Chk1 pathway. We examined the amount of Rad17 using an exogenous flag-Rad17 protein, as our anti-Rad17 antibody did not detect endogenous Rad17. Exposure of COS-1 cells to 20 or 40 μM MeHg decreased the amount of Rad17 to 87% and 37%, respectively (Figure 4). We also examined the effect of MeHg on Rad17 K/R359‒363A mutant that shows resistance to proteasomal degradation [14]. The amount of Rad17 K/R359‒363A mutant was also reduced after MeHg exposure (data not shown). In our previous work, flag-Rad17 wild type and K/R359‒363A mutant showed a half-life of 7.9 and 34.7 hours, respectively [14]. Given that MeHg exposure reduced the amount of Rad17 within 5 hours, it is likely that the decrease in Rad17 was due to protein degradation and not the inhibition of transcription or translation. These findings suggest that MeHg exposure inactivates the ATR pathway through Rad17 degradation. Moreover, as the exposure to 10 μM MeHg increased the amount of Rad17 (Figure 4), MeHg-induced cellular stress may upregulate Rad17 before degradation.
We also examined the effect of Z-VAD-FMK, a pan-caspase inhibitor, on the MeHg-induced Rad17 degradation. However, Z-VAD-FMK did not prevent Rad17 degradation (data not shown). The mechanism underlying the degradation of Rad17 remains to be elucidated.
COS-1 cells were irradiated with 30 J/m2 UV-C, and the cells were recovered for 5 hours in the presence of caffeine (A, B) or MeHg (C, D). SDS-lysate was prepared and probed with the indicated antibodies. Graphs represent results from two (B, n = 3) or three (D) independent experiments. Ratios of Chk1-pS345/Hsc70 are shown (B, D). P values were calculated using Student’s or Welch’s t-test. ***, p < 0.001.
MeHg is classified by the International Agency for Research on Cancer as a Group 2B compound, indicating its potential carcinogenicity in humans. MeHg exposure has been shown to induce DNA strand breaks [29-33], mutations [34], and chromosomal abnormalities [32] in cellular models. The possible mechanisms include direct oxidation of the DNA and inhibition of DNA repair machinery. In this study, we have demonstrated that acute exposure to MeHg inhibits the ATR pathway (Figure 3) and also induces DSBs (Figure 2). Our findings suggest that that the inhibition of ATR-dependent DNA damage responses may also be a possible mechanism underlying HeMg-induced DNA strand breaks and mutations.
We examined the effect of acute MeHg exposure on the ATR pathway several hours later. Despite a decrease in the MTS score to 7%, the expression of proteins remained unaffected (Figures 2‒4). The MTS assay monitors mitochondrial activity, and it has been demonstrated that MeHg inhibits mitochondrial function [35]. We speculate that MeHg suppresses mitochondrial function before the onset of cell death. Previous studies have shown that exposure to MeHg can cause chromosome abnormalities in experimental animals [36, 37]. Under physiological conditions, chronic exposure to MeHg may inhibit the ATR pathway, resulting in the accumulation of chromosome abnormalities observed in experimental animals.
Previous studies have reported that the ATR pathway prevents apoptotic responses upon replication stress and UV irradiation [38, 39]. ATR also plays a direct anti-apoptotic role in mitochondria [40]. Our results show that MeHg exposure leads to Rad17 degradation (Figure 4), which inactivates the ATR pathway (Figure 3). In the presence of replication stress, the ATR pathway stabilizes stalled replication forks. However, the inactivation of ATR can cause stalled replication forks to collapse, leading to the formation of DSBs, which are the most cytotoxic type of DNA lesion [1, 2] (Figure 5). Because exposure to MeHg caused replication stress and DSBs by deactivating ATR (Figures 2, 3), the inactivation of ATR and the presence of DSBs contributed to cell death, in addition to other cytotoxic effects of MeHg.
Rad17 is an important activator and regulator of the ATR pathway. Our results revealed a novel connection between acute MeHg exposure and the ATR pathway. Cell cycle progression resumes after DNA damage repair, which is not a spontaneous but an actively regulated process that involves the inactivation of DNA damage checkpoints. Our findings also suggest the presence of an unidentified mechanism of Rad17 degradation leading to the inactivation of the ATR pathway.
(A) COS-1 cells were transfected with flag-Rad17. Forty-eight hours after transfection, the cells were exposed to 10‒40 μM MeHg for 6 hours. SDS-lysate was prepared and probed with the indicated antibodies. All lanes were obtained from the same blot.
(B) The graph represents results from more than three independent experiments. Rad17 protein amount is shown as the ratio of FLAG/Hsc70. P values were calculated using Student’s t-test. *, p < 0.05. ***, p < 0.001.
Thymidine exposure, which induces replication stress, and UV irradiation activate the ATR pathway, leading to the stabilization of stalled replication forks. If the ATR pathway is inactivated, the stalled replication forks collapse and are converted into DSBs, which activates the ATM pathway. In the diagram depicted, exposure to MeHg degrades Rad17, which in turn prevents ATR activation.
We thank all the past and present members of the Laboratory of Toxicology and Environmental Health. This work was supported in part by Grants-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology [19K07079 (Y.F.), 21H04920 (Y.O.), 19H05772 (Y.O.), 22K15260 (Y.T.), 22K05345 (N.S.)].
Y. F.: conceptualization, methodology, formal analysis, investigation, resources, writing—original draft, visualization, project administration, funding acquisition. D. W.: formal analysis, investigation, visualization. Y. T. and N. S.: resources, supervision, funding acquisition. Y. O.: resources, writing—review and editing, supervision, project administration, funding acquisition.
The authors declare no conflict of interest associated with this manuscript.
