The Mre11 complex is required for ATM activation and the G2/M checkpoint
2003; Springer Nature; Volume: 22; Issue: 24 Linguagem: Inglês
10.1093/emboj/cdg630
ISSN1460-2075
Autores Tópico(s)Cell death mechanisms and regulation
ResumoArticle15 December 2003free access The Mre11 complex is required for ATM activation and the G2/M checkpoint Christian T. Carson Christian T. Carson Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Search for more papers by this author Rachel A. Schwartz Rachel A. Schwartz Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Search for more papers by this author Travis H. Stracker Travis H. Stracker Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Caroline E. Lilley Caroline E. Lilley Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Darwin V. Lee Darwin V. Lee Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Matthew D. Weitzman Corresponding Author Matthew D. Weitzman Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Christian T. Carson Christian T. Carson Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Search for more papers by this author Rachel A. Schwartz Rachel A. Schwartz Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Search for more papers by this author Travis H. Stracker Travis H. Stracker Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Caroline E. Lilley Caroline E. Lilley Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Darwin V. Lee Darwin V. Lee Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Matthew D. Weitzman Corresponding Author Matthew D. Weitzman Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Christian T. Carson1,2, Rachel A. Schwartz1,2, Travis H. Stracker1,2,3, Caroline E. Lilley1, Darwin V. Lee1 and Matthew D. Weitzman 1 1Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, 92037 USA 2Graduate Program, Department of Biology, University of California, San Diego, CA, 92093 USA 3Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6610-6620https://doi.org/10.1093/emboj/cdg630 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 G2/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. Introduction A central player in the cellular response to DSBs is the Mre11 complex, consisting of Mre11, Rad50 and NBS1 (Xrs2 in yeast) (Petrini, 1999; D'Amours and Jackson, 2002). Hypomorphic mutations in the genes for Mre11 and NBS1 result in the human genetic instability diseases ataxia-telangiectasia like disorder (A-TLD) and Nijmegen breakage syndrome (NBS), respectively (Carney et al., 1998; Stewart et al., 1999). Cell lines derived from these patients are sensitive to ionizing radiation (IR) and exhibit radioresistant DNA synthesis (RDS). Cytologic and genetic evidence suggests that the Mre11 complex acts as a DNA damage sensor (Mirzoeva and Petrini, 2001; Usui et al., 2001), but definitive evidence in mammalian cells has thus far remained elusive. The related PI3-like kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), are signal transducers of the DNA damage response. Mutations in ATM lead to ataxia-telangiectasia (A-T), and cells from these patients are hypersensitive to DSBs and defective for induction of the G1/S, intra-S and G2/M cell-cycle checkpoints (Shiloh, 2003). Null mutations of ATR are lethal, but the protein has been studied using a conditional knockout allele or dominant-negative kinase dead mutants, which sensitize cells to all forms of DNA damage and affect the intra-S and G2/M checkpoint responses (Cliby et al., 1998; Nghiem et al., 2001; Brown and Baltimore, 2003). A multitude of DNA repair and checkpoint proteins have been identified as substrates for ATM and ATR kinase activity, including the checkpoint kinases Chk1 and Chk2, as well as 53BP1 (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002), H2AX (Paull et al., 2000), BRCA1, p53 and RPA32 (reviewed in Kastan and Lim, 2000; Shiloh, 2003). Both Mre11 and NBS1 are also targets of ATM and possibly ATR (Gatei et al., 2000; Lim et al., 2000; Wu et al., 2000; Zhao et al., 2000; Costanzo et al., 2001; D'Amours and Jackson, 2001). Recent studies have begun to elucidate the cellular requirements for the activation of ATM and ATR upon DNA damage. It has been proposed that ATM is activated by intermolecular autophosphorylation on Ser1981 (Bakkenist and Kastan, 2003). In the case of ATR it has been suggested that RPA bound to single-stranded DNA is required for recruitment of ATR to sites of DNA damage (Zou and Elledge, 2003). The Mre11 complex has been proposed to facilitate ATM activation and functionally interact with ATR (Usui et al., 2001; Williams et al., 2002), but knowledge of molecular mechanisms to support these links is lacking in mammalian cells. We have demonstrated that adenovirus (Ad) avoids detrimental genome concatemerization through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins (Stracker et al., 2002). Here we show for the first time that infection with an adenovirus lacking the E4 region induces a cellular DNA damage response that requires ATM and ATR. We identified mutants of E1b55K that can selectively degrade different substrates. Using these E1b55K mutants we show that the Mre11 complex is required for the activation of ATM and ATR in response to Ad infection. In addition, we show that Mre11 degradation abrogates ATM autophosphorylation and signaling as well as the ATM-dependent G2/M checkpoint in response to DSBs. Together, these results support a role for the mammalian Mre11 complex in sensing DSBs and adenoviral infection. Results E4-deleted adenovirus elicits a cellular DNA damage response Infection with an E4-deleted Ad results in end-joining of viral genomes by cellular factors and this is accompanied by hyperphosphorylation of NBS1, which is indicative of a cellular DNA damage response (Stracker et al., 2002). To investigate this further, we examined whether other damage-response proteins become phosphorylated upon infection with the E4-deleted Ad dl1004 (Figure 1A). Immunoblotting of cell lysates over a time-course of infection revealed slower migrating forms of BRCA1 and RPA32. Antibodies that recognize specific phosphorylated residues demonstrated phosphorylation of Chk1, Chk2, 53BP1, p53 and ATM. The p53 protein also appeared to be stabilized, which may be due to E1a interference with proteasomes or the E6 protein expressed in the HeLa cells. These results indicate that infection with an E4-deleted virus leads to ATM activation and signaling to proteins involved in the cellular DNA damage response. Figure 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. Download figure Download PowerPoint These phosphorylation events were not detected to a significant level during infection with wild-type Ad (Figure 1B), suggesting that the wild-type virus can prevent activation of this cellular response. At late time points we observed slight activation of ATM and phosphorylation of 53BP1 and Chk2 (data not shown), but these signals were significantly lower than those observed during infection with the E4-deleted virus. Many of the phosphorylation events that we examined were on substrates of the PI3-like kinases, ATM, ATR and DNA-PKcs, and therefore we tested whether the virus indirectly affects the activities of these kinases by altering their steady-state levels (Figure 1B). We did not observe any alteration in the levels of ATM, ATR and DNA-PKcs over a 24 h time-course of infection with wild-type adenovirus type 5 (Ad5). In contrast, the Mre11 complex was degraded as the infection progressed. We next sought to determine the roles of ATM and ATR in the DNA damage response to E4-deleted adenovirus. Phosphorylation of substrates during infection with the E4-deleted virus dl1004 was investigated in cells deficient for ATM or ATR kinase activity. In ATM-deficient cells, phosphorylation of 53BP1 on Ser25 was abrogated, but all other phosphorylation events were detected (Figure 2A). These events were ablated by 5 mM caffeine, a dose that inhibits the kinase activity of both ATM and ATR in vitro (Sarkaria et al., 1999). These data suggest that while ATM signaling was not absolutely required, a caffeine-sensitive kinase was involved. We also performed the infections in A-T cells in the presence of 20 and 150 μM wortmannin (Figure 2B), which inhibits ATM and DNA-PKcs at the lower concentration and ATR at the higher concentration. We observed that treatment with 150 μM, but not 20 μM wortmannin prevented the phosphorylation events, suggesting that ATR may be involved in this signaling. The role of ATR was further examined using a cell line that expresses a doxycycline-inducible trans-dominant negative mutant (Nghiem et al., 2001). Infection with the E4-deleted virus led to phosphorylation of protein substrates in control cells (ATR-WT), but induction of the trans-dominant protein (ATR-KD) prevented phosphorylation of Chk1 on Ser345, and blocked hyperphosphorylation of RPA32 (Figure 2C). These phosphorylation events were not detected in either cell line during infection with wild-type Ad. These results suggest that ATM and ATR or another kinase sensitive to both caffeine and wortmannin may be redundant for the phosphorylation of damage response proteins during viral infection. Figure 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. Download figure Download PowerPoint Many proteins involved in the DNA damage response accumulate in foci at sites of DSBs (Rouse and Jackson, 2002). In a similar fashion, the Mre11 complex accumulates at foci surrounding viral replication centers formed during infection with E4-deleted adenovirus (Stracker et al., 2002). Using immunofluorescence staining we examined whether other damage proteins also accumulated at viral centers during infection. Viral replication centers were identified with an antibody to the viral DNA binding protein (DBP), and we observed foci of BRCA1, 53BP1 and γ-H2AX (the phosphorylated form of H2AX) at these discrete sites (Figure 3A). We also examined localization of the ATM and ATR kinases (Figure 3B). Staining with a phosphospecific antibody to Ser1981 of ATM demonstrated accumulation of activated ATM at E4-deleted (dl1004) viral centers. This suggests that ATM is activated in the absence of E4 and accumulates at sites of virus replication. In contrast, ATM autophosphorylation was not observed in Ad5-infected cells (Figure 3B), although some staining was visible at late stages of infection (data not shown). Infection with both wild-type Ad5 and the E4-deleted virus resulted in accumulation of ATR, ATRIP and RPA at viral replication centers (Figure 3B and data not shown). These data suggest that ATR localization is independent of activation of the DNA damage response and the Mre11 complex (as the complex is degraded in wild-type Ad5 infection). Figure 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). Download figure Download PowerPoint We examined whether accumulation of the Mre11 complex at viral centers was dependent on the kinase activities of ATM/ATR. A-T cells were infected with the E4-deleted virus in the presence or absence of 5 mM caffeine. Recruitment of Rad50 to foci at viral replication centers occurred even after caffeine treatment of A-T cells (Figure 3C). We also examined the formation of γ-H2AX foci at viral centers (Figure 3D). Foci of γ-H2AX were observed at viral centers in A-T cells, but in contrast to Rad50 foci, the accumulation of γ-H2AX was abrogated in the presence of caffeine. This indicates that foci formation by the Mre11 complex is resistant to caffeine and is independent of γ-H2AX foci, consistent with previous reports showing that accumulation of the Mre11 complex at sites of damage is independent of ATM and γ-H2AX in response to DSBs (Mirzoeva and Petrini, 2001; Celeste et al., 2003). Together, these data suggest that the Mre11 complex can function upstream of, or coincident with, ATM and ATR activation. It has previously been demonstrated that the ATM protein becomes associated with chromatin upon DNA damage (Andegeko et al., 2001). This was shown using a biochemical fractionation procedure in which a portion of the ATM pool was found to be resistant to detergent extraction after treating cells with agents that lead to DSBs (Andegeko et al., 2001). We used this assay to assess whether ATM retention also occurred during virus infection (supplementary figure 1, available at The EMBO Journal Online). Cells infected with either wild-type Ad5 or the E4-deleted virus dl1004 were harvested and fractionated by successive detergent extractions. Immunoblotting of cell-equivalent aliquots showed that the amount of loosely bound ATM removed by the first detergent extraction (FI) was similar between the samples. However, infection with the E4-deleted virus but not wild-type Ad5 led to a portion of the ATM protein being retained in fraction III (FIII). This assay supports the conclusion that infection with an E4-deleted adenovirus elicits a DNA damage response. E1b55K/E4orf6 target the Mre11 complex for degradation The data thus far demonstrated that removal of the Mre11 complex by wild-type adenovirus correlated with prevention of the DNA damage response to infection. We further characterized the mechanism and specificity of Mre11 complex degradation by the viral E1b55K/E4orf6 proteins. In order to study degradation induced by E1b55K and E4orf6 in multiple cell lines, we expressed them individually using replication-deficient, recombinant, E1-deleted adenovirus vectors (rAd.E1b55K and rAd.E4orf6). Degradation of Mre11, Rad50 and NBS1 proteins was only observed in cells infected with both vectors, whereas either vector alone had minimal effect (Figure 4A). Degradation of p53 was also observed, as previously reported (Cathomen and Weitzman, 2000; Querido et al., 2001a). These results show that the E1b55K/E4orf6 complex is sufficient for inducing degradation. Figure 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. Download figure Download PowerPoint The E1b55K/E4orf6 complex has been shown to recruit cellular factors and function as a von Hippel-Lindau (VHL)-like ubiquitin ligase to mediate polyubiquitination of the p53 protein in vitro (Querido et al., 2001a; Harada et al., 2002). In the case of p53 it has been demonstrated that degradation requires E1b55K binding to the N-terminus (Cathomen and Weitzman, 2000; Querido et al., 2001b). Therefore we explored whether there is an interaction between E1b55K and the Mre11 complex. Immunoprecipitation of E1b55K from cells infected with rAd.E1b55K was followed by immunoblotting, and revealed pull-down of the Mre11, Rad50 and NBS1 proteins but not ATM (Figure 4B and data not shown). The reciprocal immunoprecipitations of NBS1 and Rad50 also pulled down E1b55K (Supplementary figure 2). These data demonstrate that the E1b55K protein interacts with the Mre11 complex. Degradation and substrate recognition was investigated further by screening a panel of viruses that express mutant forms of the E1b55K protein (R.A.S. and M.D.W., unpublished data). The E1b55K viruses that we tested harbor point mutations, linker insertions or large deletions, and have previously been well characterized for growth (Yew et al., 1990; Shen et al., 2001). We examined the effect of infection with these mutant viruses on the levels of p53 and Mre11 and compared them with infection with wild-type Ad5 and the E1b55K-deleted virus dl110 that does not degrade either protein. We identified two mutants that showed differential substrate specificity for Mre11 or p53 (Figure 4C). The E1b55K H354 mutant (Yew et al., 1990) degrades p53 but not the Mre11 complex, while the E1b55K R240A virus (Shen et al., 2001) degrades the Mre11 complex but not p53, demonstrating that E1b55K is involved in substrate recognition and that specificity for the Mre11 complex or p53 is separable. In order to study the effects of the mutant E1b55K proteins on signaling events in the absence of other viral proteins, we generated stable HeLa and U2OS cell lines expressing each mutant E1b55k protein from retrovirus vectors. The E1b55K proteins were stably expressed at similar levels in these cell lines (Figure 5A) and immunofluorescence revealed the wild-type and mutant E1b55K proteins at speckles in the cytoplasm (Figure 5B), consistent with previous reports of E1b55K localization (Zantema et al., 1985). Co-staining showed that the localization and levels of NBS1 were unaffected by E1b55K expression in these cells (Figure 5B). Co-expression of E4orf6 results in nuclear accumulation of E1b55K (Goodrum et al., 1996; Dobbelstein et al., 1997; Cathomen and Weitzman, 2000). The stable cell lines were infected with the rAd.E4orf6 virus, and in each case the E1b55K protein was found relocated to the nucleus in a predominantly diffuse pattern (Figure 5B). This indicates that the wild-type and mutant proteins all interact with E4orf6, consistent with published data from immunoprecipitation experiments (Rubenwolf et al., 1997; Shen et al., 2001). In the case of wild-type E1b55K and the R240A mutant, E4orf6 expression and nuclear accumulation were accompanied by degradation of Mre11, Rad50 and NBS1 as revealed by both immunofluorescence and immunoblotting (Figure 5B and C). The steady-state levels of members of the Mre11 complex were unaffected in cells expressing the H354 mutant or in a control cell line expressing green fluorescent protein (GFP). Similar results were observed for both the U2OS- and HeLa-derived cell lines. These cell lines thus provide a useful and controlled system to study cellular processes in the absence of the Mre11 complex. Figure 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. Download figure Download PowerPoint Mre11 degradation prevents ATM and ATR signaling in response to viral infection We tested whether degradation of the Mre11 complex was essential in order to prevent the cellular damage response to virus infection. The U2OS cell lines expressing GFP or the E1b55K proteins were infected with an adenovirus mutant (dl1016) that was deleted for E1b55K but expressed functional E4orf6 (Figure 5C). This virus was used because it has an additional deletion in E4orf3, a gene that we previously showed to mislocalize and inactivate the Mre11 complex (Stracker et al., 2002). Infection of the GFP control cells and those expressing the H354 mutant did not result in degradation of Mre11, Rad50 and NBS1, and was accompanied by phosphorylation of ATM and ATM/ATR substrates, including NBS1, BRCA1, RPA32, Chk1 and 53BP1. Infection of cells expressing wild-type E1b55K (WT) and the R240A mutant resulted in degradation of the Mre11 complex, and there was no evidence of ATM activation or the phosphorylation of ATM/ATR substrates after dl1016 infection. This shows that targeted degradation of the Mre11 complex, and not p53, is important to prevent activation of ATM/ATR in response to viral infection. Similar results were observed for the HeLa-derived cell lines (data not shown). Mre11 degradation prevents ATM activation and signaling in response to DSBs We hypothesized that by degrading the Mre11 complex, adenoviral E1b55K/E4orf6 proteins could prevent activation of signaling cascades induced by other forms of exogenous DNA damage. We therefore tested the effect of E1b55K/E4orf6 proteins on the cellular DNA damage response to IR. Cells expressing GFP or E1b55K proteins were infected with the recombinant vector rAd.E4orf6, prior to treatment with 10 Gy of IR. Expression of E4orf6 in cells containing wild-type E1b55K or the R240A mutant led to the degradation of the Mre11 complex, accompanied by greatly reduced ATM autophosphorylation and decreased phosphorylation of the ATM substrates BRCA1, Chk2 and 53BP1 (Figure 6A). In contrast, E4orf6 expression in cells containing the H354 mutant failed to degrade the Mre11 complex and had no effect on the damage response. Similar results were observed in the HeLa-derived cell lines (data not shown). In addition, foci of autophosphorylated ATM were detected after IR, but were absent with prior degradation of the Mre11 complex by E1b55K/E4orf6 (Figure 6B). These results suggest that the Mre11 complex is required for ATM activation and signaling after DSBs and that E1b55K/E4orf6 prevents the response by targeting the Mre11 complex for degradation. Figure 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. Download figure Download PowerPoint Mre11 degradation abrogates the ATM-dependent early G2/M checkpoint ATM signaling has been shown to be required for the early G2/M checkpoint in response to IR (Xu et al., 2002). This transient checkpoint is activated in cells that were in G2 when they incurred damage and prevents their progression into mitosis (M). Cells with functional ATM are prevented from entering M within the first 30–90 min after IR. However, ATM is not required for prolonged G2 accumulation after IR (Xu et al., 2002). There have been conflicting reports about the role of the Mre11 complex in the early G2/M checkpoint (Buscemi et al., 2001; Xu et al., 2001, 2002; Williams et al., 2002). We reasoned that if the Mre11 complex was required for ATM activation and signaling, cells expressing E1b55K/E4orf6 should exhibit a defect in the ATM-dependent G2 arrest after IR. In the absence of E4orf6, this checkpoint was intact in HeLa cells expressing either GFP or any of the E1b55K proteins (Figure 6C). However, in the wild-type E1b55K cell line infected with rAd.E4orf6, this checkpoint was abrogated, and cells continued to enter M phase after IR treatment. This was also seen in cells expressing the R240A mutant, but not in the GFP or H354 cell lines infected with rAd.E4orf6. These results show that the Mre11 complex is required for the early G2/M checkpoint, possibly via ATM activation. Mre11 degradation by E1b55K/E4orf6 did not abrogate ATM-independent long-term G2 accumulation after IR (data not shown). To demonstrate that the effect o
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