The Mre11 complex is required for ATM activation and the G2/M checkpoint

Abstract

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.

Fig. 1. Infection with the E4-deleted virus dl1004 activates signaling pathways involved in the cellular DNA damage response, but does not affect the steady-state levels of PI3-like kinases. (A) HeLa cells were mock infected (M) or infected with dl1004 for the indicated times and harvested for immunoblotting. (B) HeLa cells were mock infected (M), infected with dl1004 or Ad5 for the indicated times and harvested for immunoblotting.

Fig. 2. ATM and ATR are activated by infection with E4-deleted adenovirus. Phosphorylation of cellular proteins was examined by immunoblotting of lysates from A-T cells (GM5849) (A and B) and cells expressing inducible ATR proteins (C), either wild-type (WT) or kinase-dead (KD) after doxycycline treatment. In each case cells were uninfected (M), infected with wild-type Ad5 or infected with the E4-deleted virus dl1004 (ΔE4). The infections in A-T cells were also performed in the presence of 5 mM caffeine (A) or 20 µM and 150 µM wortmannin (B) throughout infection. A-T cells were infected for 30 h and U2OS cells expressing ATR were infected for 24 h. Ku70 and Ku86 served as loading controls.

Fig. 3. DNA repair proteins accumulate at sites of viral replication during infection with a virus lacking E4. (A) U2OS cells were untreated (Mock) or infected with the E4-deleted virus dl1004 (m.o.i. = 25), and proteins were visualized at 23 h.p.i. by immunofluorescence. Viral replication centers were localized by staining for DBP. (B) Localization of kinases during virus infection. U20S cells were infected with wild-type Ad5 or dl1004, and stained with antibodies specific to the autophosphorylated site at S1981 of ATM or ATR. (C) Formation of Rad50 foci at viral replication centers is independent of signaling events and is caffeine resistant. The A-T cells were infected with the dl1004 virus in the presence and absence of 5 mM caffeine and harvested 30 h.p.i. (D) Foci of γ-H2AX at viral replication centers are sensitive to caffeine and are not required for Rad50 foci. The A-T cells were infected with the dl1004 virus as in (C).

Fig. 4. The adenoviral E1b55K protein is important for substrate recognition and degradation of the Mre11 complex. (A) The E1b55K and E4orf6 proteins are required for degradation of the Mre11 complex. U2OS cells were untreated or infected with E1-deleted recombinant Ad vectors expressing E1b55K and E4orf6 alone or in combination (m.o.i. = 10 and 50 p.f.u., respectively). Cells were harvested at 30 h.p.i. for immunoblotting. Ku86 served as a loading control. (B) The E1b55K protein interacts with the Mre11 complex. U2OS cells were either untreated (Mock) or infected with rAd.E1b55K for 15 h. Cell lysates were immunoprecipitated with an antibody to E1b55K or a control antibody to adenovirus DBP. Immunoblotting is shown for the lysates (5% of input) or the precipitates. (C) Mutations in E1b55K separate degradation of p53 and the Mre11 complex. U2OS cells were infected with wild-type Ad5, a virus deleted of E1b55K (dl110) and two viruses expressing mutant E1b55K proteins. Cells were harvested at the indicated times for immunoblotting. Ku70 served as a loading control.

Fig. 5. Degradation of the Mre11 complex prevents activation of the cellular DNA damage response during adenovirus infection. (A) Stable cell lines expressing E1b55K proteins. The E1b55K coding regions from wild-type and mutant viruses were cloned into retrovirus vectors and used to make stable cell lines. A GFP-expressing retrovirus was used as a control. Expression of E1b55K in HeLa and U2OS-derived cell lines was confirmed by immunoblotting. Ku70 served as a loading control. (B) E4orf6 recruits E1b55K into the nucleus and NBS1 is degraded. The U2OS cell lines described in (A) were uninfected (upper panels) or infected with rAd.E4orf6 (lower panels), and immunostained with antibodies to E1b55K and NBS1. Similar results were seen in the HeLa-derived cell lines. (C) Immunoblotting reveals that degradation of the Mre11 complex prevents cellular responses to viral infection. Cell lines derived from U2OS that express GFP or E1b55K proteins were infected with the virus dl1016 that is mutated for both E1b55K and E4orf3 genes. Infection of the GFP cell line with Ad5 served as a positive control. Cells were harvested at 30 h.p.i.

Fig. 6. Degradation of the Mre11 complex by E1b55K/E4orf6 prevents the cell from activating a DNA damage response and the G₂/M checkpoint after IR. (A) Degradation of the Mre11 complex prevents ATM activation and signaling upon IR. The stable U2OS cells lines were infected with rAd.E4orf6 for 24 h prior to 10 Gy IR, and were harvested for immunoblotting after a further 2 h. (B) Degradation of Mre11 prevents formation of autophosphorylated ATM foci in response to IR. Cells were either uninfected or infected with rAd.E4orf6, and then exposed to irradiation 1 h before immunofluorescence. (C) Degradation of the Mre11 complex abrogates the ATM-dependent early G₂/M checkpoint in response to IR. Representative flow cytometry profiles of rAd.E4orf6-infected cells 1 h post-treatment, with or without 10 Gy IR. Cells were stained for DNA content (x-axis) and histone H3 phosphorylation (y-axis). The population of cells in mitosis is encircled and its percentage of the total cells is indicated. The change in the number of mitotic cells 1 h after IR is indicated in the graph below, in which the number of mitotic cells is presented as a percentage of the number detected in unirradiated samples of the same condition. The mean and standard deviation are shown for the results of three independent experiments for each condition.

Fig. 7. Degradation of the Mre11 complex prevents ATM activation and retention on chromatin in response to agents that cause DSBs. (A) Proteasome-mediated degradation is required for E1b55K/E4orf6 to prevent ATM activation. U2OS cells expressing wild-type E1b55K (U2OS-E1b.WT) were either mock infected (M) or infected with rAd.E4orf6 for the indicated times (h). Cells were irradiated with 10 Gy and harvested at 1 h post-treatment. This experiment was performed in parallel in the presence or absence of the proteasome inhibitors, MG132 (10 µM) and epoxomicin (1 µM). Inhibitors were added 2 h.p.i. and left on for the duration of the infection. (B) E1b55K/E4orf6 prevent ATM autophosphorylation in response to agents that lead to DSBs. U2OS cells expressing wild-type E1b55K (U2OS-E1b.WT) were either uninfected (–) or infected (+) with rAd.E4orf6. At 24 h.p.i. these cells were either untreated (Mock) or treated with 50 ng/ml neocarzinostatin (NCS) for 4 h, 1 µM camptothecin (CPT) for 2 h, or 2 mM hydroxyurea (HU) for 4 h. (C) Degradation of the Mre11 complex prevents ATM retention after treatment with NCS. HeLa cells expressing wild-type E1b55K (HeLa-E1b.WT) were either mock infected (Mock) or infected with rAd.E4orf6. At 24 h.p.i. the cells were treated with 200 ng/ml NCS for 30 min. The cells were harvested and biochemically fractionated as described in Materials and methods. The intensity of bands was quantitated and the ratios of ‘sample without NCS treatment’ to ‘sample with NCS’ are given below. Immunoblotting for Mre11 demonstrated degradation, and blotting for E1b55K served as an internal control for equal loading.

Fig. 8. Complementation of A-TLD1 cells with Mre11 cDNA restores ATM activation and signaling in response to IR. (A) ATM activation and signaling is abrogated in A-TLD1 cells and can be restored by complementation with wild-type Mre11. Cells were either infected with virus (m.o.i. = 100) or exposed to IR. Cells were harvested 24 h.p.i. with wild-type Ad or the E4-deleted virus (ΔE4). Irradiated cells were harvested at 45 min after recovery from exposure to the indicated amount of IR. (B) Model for the role of the Mre11 complex in DNA damage response to virus infection and DSBs. Infection with an E4-deleted adenovirus leads to activation of ATM and ATR. These kinases are redundant for the phosphorylation of many substrates, including NBS1. However, these kinases also have specific targets, such as Ser25 on 53BP1 for ATM, and Ser345 on Chk1 for ATR. Degradation of the Mre11 complex by E1b55K/E4orf6 proteins prevents the activation of the ATM/ATR kinases. The Mre11 complex also plays a role as an upstream sensor in response to DSBs, and degradation of the Mre11 complex prevents ATM autophosphorylation and signaling, in addition to the ATM-dependent G₂/M checkpoint.