ATM induces MacroD2 nuclear export upon DNA damage

Abstract

ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of specific transferases and hydrolases. However, except for the transferase PARP1/ARDT1 little is known about regulation of these enzymes. We found that MacroD2, a mono-ADP-ribosylhydrolase, is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. We show that the export is dependent on the phosphorylation of two SQ/TQ motifs, suggesting a novel direct interaction between ATM and ADP-ribosylation. Lastly, we show that MacroD2 nuclear export temporally restricts its recruitment to DNA lesions, which may decrease the net ADP-ribosylhydrolase activity at the site of DNA damage. Together, our results identify a novel feedback regulation between two crucial DNA damage-induced signaling pathways: ADP-ribosylation and ATM activation.

Figure 1.

MacroD2 exports from the nucleus upon DNA damage. (A) Microirradiation live-cell microscopy of U2OS cells stably expressing the mEGFP-MacroD2 full–length construct. Arbitrary medium and high laser energy values are used to induce different amounts of damage to the cell. Scale bar, 10 μm. (B) Quantification of A. (C) Live-cell imaging of U2OS-mEGFP-MacroD2 full-length cells treated either with etoposide 10 μM or DMSO. Scale bar, 10 μm. (D) Quantification of C. (E) Immunocytochemistry with anti-MacroD2 antibody on cells treated either with etoposide 10 μM or DMSO. Scale bar, 10 μm. (F) Quantification of E in three independent experiments. Statistics performed with unpaired non-parametrical Kolmogorov-Smirnov test. (G) Predictions of MacroD2 secondary structure. Sequence of MacroD2 Isoform 1 of Uniprot was analysed with the online software PredictProtein (33). The first line shows prediction for the presence of helices or strands of beta sheets; the second line shows prediction for each residue to be exposed or buried; the third line shows prediction of disorder. In B and D, 50–100 cells were quantified from three independent experiments. Error bars are 95% confidence interval. See also Supplementary Figure S1.

Figure 2.

ATM kinase activity induces MacroD2 nuclear export. (A) Microirradiation live-cell microscopy of U2OS cells stably expressing mEGFP-MacroD2 full-length construct and treated with Olaparib 2 μM or DMSO. The focus of laser microirradiation is indicated with a yellow circle. Scale bar, 10 μm. (B) Quantification of A. (C) Microirradiation live-cell microscopy of U2OS cells stably expressing mEGFP-MacroD2 full-length construct and treated with KU55933 10 μM, VE-821 1 μM, NU7441 1 μM or DMSO. Scale bar, 10 μm. (D) Quantification of C. (E) Quantification of live-cell imaging experiments with U2OS cells stably expressing mYFP-MacroD2 C-terminal domain, pre-treated with siRNA against ATM or negative control siRNA for three days and treated with etoposide 10 μM. Error bars are 95% confidence interval. One representative experiment with more than 200 cells is shown. (F) Autoradiography and Coomassie staining of mEGFP-MacroD2 full-length protein purified from stably-expressing U2OS cell lysate, previously treated or not with benzonase and spiked with (³²P)γ-ATP. (G) Autoradiography and Coomassie staining of mEGFP-MacroD2 macrodomain or mEGFP-MacroD2 C-terminal region proteins immunopurified from stably-expressing U2OS cell lysates, previously treated with benzonase and spiked with (³²P)γ-ATP. (H) Coomassie and autoradiography of mEGFP-MacroD2 C-terminal domain added to HEK293 cell lysate, treated or not with benzonase and KU55933, spiked with (³²P)γ-ATP and successively immunopurified. (I) Quantification of H IP results in three independent experiments. Error bars, SEM. (J) Abundance of phosphorylated peptides of mEGFP-MacroD2 immunopurified from U2OS stably expressing the protein and treated with etoposide 10 μM or DMSO. Quantification of three independent experiments. Error bars, SEM. Statistics performed with paired t-Test with two-tailed distribution. In B and D, 50–100 cells were quantified from three independent experiments. Error bars are 95% confidence interval. See also Supplementary Table S1.

Figure 3.

Sequence requirements for the phosphorylation are consistent with the direct involvement of ATM. (A) Schematic view showing the four SQ motifs present on the C-terminal domain. (B) Microirradiation live-cell microscopy of HeLa cells transfected with mEGFP-MacroD2 full-length or mEGFP-MacroD2 S276,345,415,426A constructs. Scale bar, 10 μm. (C) Quantification of B. (D) Quantification of microirradiation live-cell microscopy of HeLa cells transfected with mEGFP-MacroD2 full length or the different serine triple mutants (Examples shown in Supplementary Figure S3A). (E) Coomassie and autoradiography of GST-mEGFP-MacroD2 (aa382–418) wild-type and His-mEGFP-MacroD2 (aa382–418) (wild-type or mutant proteins) immunopurified from HEK293 cell lysate, treated with benzonase and spiked with (³²P)γ-ATP. (F) Quantification of E in three independent experiments. Error bars, SEM. Statistics performed with unpaired non-parametrical Mann-Whitney test against the wild-type. *P ≤ 0.05; n.s. is P > 0.05. (G) Microirradiation live-cell microscopy of HeLa cells transfected with mEGFP-MacroD2 (aa382–418) wild-type construct or mutations of S415A or Q416A. Scale bar, 10 μm. (H) Quantification of G. In C, D and G, 50–100 cells were quantified from three independent experiments. Error bars are 95% confidence interval. See also Supplementary Figure S3.

Figure 4.

ATM activity regulates the recruitment of MacroD2 to sites of DNA damage. (A) Recruitment of tagged mEGFP-MacroD2 constructs (full length, macrodomain or C-terminal domain) in HeLa cells. The focus of laser micro-irradiation is indicated with yellow arrowheads. Scale bar, 10 μm. (B) Recruitment of tagged mEGFP-MacroD2 constructs (wild-type or S276 345 415 426A mutant) in HeLa cells. The focus of laser micro-irradiation is indicated with a yellow circle. Scale bar, 10 μm. (C) Quantification of B performed on 15 cells from three independent experiments. Error bars are 95% confidence interval.