ABSTRACT
Adenovirus (Ad) mutants that lack early region 4 (E4) are unable to produce the early regulatory proteins that normally inactivate the Mre11/Rad50/Nbs1 (MRN) sensor complex, which is a critical component for the ability of cells to respond to DNA damage. E4 mutant infection therefore activates a DNA damage response, which in turn interferes with a productive viral infection. MRN complex proteins localize to viral DNA replication centers in E4 mutant-infected cells, and this complex is critical for activating the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which phosphorylate numerous substrates important for DNA repair, cell cycle checkpoint activation, and apoptosis. E4 mutant growth defects are substantially rescued in cells lacking an intact MRN complex. We have assessed the role of the downstream ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. We did not identify a role for either ATM or ATR in “repair” of E4 mutant genomes to form concatemers. ATR was also not observed to contribute to E4 mutant defects in late protein production. In contrast, the kinase activity of ATM was important for preventing efficient E4 mutant DNA replication and late gene expression. Our results suggest that the MRN complex interferes with E4 mutant DNA replication at least in part through its ability to activate ATM.
INTRODUCTION
Adenovirus (Ad) infection delivers a linear double-stranded DNA genome to the nucleus of infected cells. This exogenous DNA has the potential to activate cellular DNA damage responses (DDRs) (reviewed in reference 1), which can impede a productive viral infection (2–5). Consequently, Ad produces early gene products that interfere with the activity of several cellular DDR proteins. Proteins from early region 1b (E1b-55kDa) and E4 orf6 (E4-34kDa) form a complex that redirects a cellular CUL5-containing E3 ubiquitin ligase to target specific proteins for ubiquitination and proteasome-mediated degradation (6–8). Mre11 of the MRN complex (9, 10), the nonhomologous end-joining (NHEJ) enzyme DNA ligase IV (11), and the tumor suppressor p53 (8) are examples of DDR proteins targeted for degradation by this complex. E4 orf3 produces an 11-kDa protein (E4-11kDa) that redistributes Mre11 to nuclear filaments (2, 10, 12), and both E4-34kDa and E4-11kDa bind and inactivate DNA-dependent protein kinase (DNAPK), which is an essential kinase for NHEJ (13). Mutants deleted for the E4 transcription unit are unable to produce these viral proteins and consequently activate a cellular DDR in infected cells (9). This response includes activation of the kinases ATM and ATR, which phosphorylate numerous downstream substrates important for repair, cell cycle arrest, and apoptosis (reviewed in reference 14). Ad infection also induces the reorganization of DDR proteins to nuclear foci that can be viewed by immunofluorescence staining. Mre11 and mediator of DNA damage checkpoint protein 1 (Mdc1) are redistributed to early foci that appear prior to the onset of viral DNA replication (4, 5). Phosphorylated ATM (pATM) is found in foci that contain input E4 mutant DNA (15). In E4 mutant-infected cells, Mre11 and pATM are present in viral replication centers that contain the 72-kDa DNA binding protein produced from E2 (E2-72kDa) (9, 10). ATR is also found to localize to viral DNA replication centers in both Ad5 and E4 mutant infections (9).
Activation of cellular DDRs severely reduces productive growth of E4 mutants in cells. Viral genomes are concatenated by DNAPK-mediated NHEJ in E4 mutant infections (13), potentially affecting virus yields because concatemers are too large to be packaged in assembling virions. However, cells that lack DNAPK and fail to concatenate viral genomes still do not rescue E4 mutant defects in viral DNA replication following low-multiplicity infections (4, 16), indicating that genome concatenation does not account for all E4 mutant growth defects. Work from several groups has shown that the MRN complex interferes with E4 mutant growth by inhibiting viral DNA replication (2–5). Cells lacking either Mre11 or Nbs1 support efficient E4 mutant replication and growth (2, 5). Knockdown of Mre11, Rad50, or Nbs1 by RNA interference also dramatically rescues the DNA replication phenotype of an E4 mutant in HeLa cells (4). The mechanism used by the MRN complex to interfere with E4 mutant DNA replication is currently being investigated. Recent results indicate that the nuclease activity of Mre11 is not critical for the DNA replication defect, suggesting that nuclease-mediated destruction of the viral origin of replication is unlikely to be the primary mechanism involved (15). However, Nbs1-dependent binding of Mre11 to viral DNA is important for inhibiting E4 mutant DNA replication (5). These observations raise the possibility that the MRN complex may be able to inhibit E4 mutant DNA replication by physically interacting with the genome and perhaps preventing viral DNA replication proteins from being able to access the origin of replication located at the termini of the linear DNA genome (5, 15). The MRN complex acts as a sensor to detect DNA damage, but it is also critical for activating signaling cascades mediated by the ATM and ATR kinases in response to DNA damage. The MRN complex could interfere with E4 mutant DNA replication as a consequence of either its DNA damage-sensing activity or its ability to stimulate DDR kinases. We have sought to more specifically address the role of the ATM and ATR kinases in limiting a productive E4 mutant infection. We did not identify a role for either ATM or ATR in E4 mutant genome concatenation; however, the kinase activity of ATM is important for inhibiting E4 mutant DNA replication.
MATERIALS AND METHODS
Cells and viruses.HeLa and E4 mutant-complementing W162 cells (17) were grown in Dulbecco's modified Eagle medium (DMEM) (Fisher) supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 10 μg/ml streptomycin. GM16666 (referred to here as ATM−) and GM16667 (referred to here as ATM+) (GM16667 cells are derived from GM16666 cells and have been complemented with a construct producing ATM) were purchased from the Coriell cell repository and maintained in DMEM supplemented with 10% FBS, 10 U/ml penicillin, 10 μg/ml streptomycin, and 100 μg/ml Hygromycin B (Invitrogen). NBS-ILB1 cells stably transduced with pLXIN retroviral vector alone (referred to here as Nbs1− cells) and NBS-ILB1 cells that were stably transduced with the pLXIN retroviral vector expressing the wild-type NBS1 protein (referred to here as Nbs1+ cells) were obtained from Pat Concannon (18) and maintained in DMEM supplemented with 10% FBS, 10 U/ml penicillin, 10 μg/ml streptomycin, and 500 μg/ml G418 (Invitrogen). Wild-type Ad5 and E4 mutant H5dl1007 (E4-) (19) were propagated on W162 cells for stock preparation. Titers were determined on W162 cells as described previously (20) and were expressed as fluorescence-forming units/ml (FFU/ml).
Immunofluorescence microscopy.Cells were grown on 12-mm-diameter cover glasses placed in culture dishes and were either left uninfected or infected with E4- or Ad5 and fixed for immunofluorescence staining at the desired time point (4). Briefly, cells were rinsed twice with phosphate-buffered saline (PBS) (0.058 M Na2HPO4, 0.017 M NaH2HPO4, 0.069 M NaCl), prefixed with 1% paraformaldehyde–PBS for 3 min, extracted with 1% Triton X-100–PBS for 15 min, and fixed with 4% paraformaldehyde in PBS for 15 min followed by three 5-min washes with PBS. Cover glasses were then stored at 4°C in PBS until use. For immunofluorescence staining, the cover glasses were rinsed in TBST (Tris-buffered saline Tween; 100 mM Tris-Cl [pH 7.5], 150 mM NaCl, 0.1% Tween 20) followed by incubation with 15 μl of blocking reagent (100 mM Tris [pH 7.5], 150 mM NaCl, 0.5% blocking reagent powder; GE Amersham) for 45 min. Cover glasses were then rinsed with TBST, and 15 μl of primary antibodies diluted in blocking reagent was added for 45 min to 1 h. Cover glasses were then washed 3 times for 10 min each time with TBS (Tris-buffered saline; 100 mM Tris-Cl [pH 7.5], 150 mM NaCl) and rinsed once with TBST. Finally, 10 μl of secondary antibodies diluted in blocking reagent was added to each cover glass. After 45 min of incubation, the cover glasses were washed 3 times for 10 min each time with TBS and mounted on a glass slide with 4 μl of Vectashield (Vector Laboratories) as the mounting medium.
The following primary antibodies were used for immunofluorescence microscopy at the dilutions specified: goat polyclonal anti-Mre11 (Santa Cruz) (1:100), mouse monoclonal B6-8 anti-72K (a gift from A. Levine) (1:100), rabbit polyclonal anti-72K (a gift from T. Linné) (1:1,500), mouse monoclonal anti-phospho-ATM (p-S1981) (Abcam) (1:20), rat monoclonal anti-bromodeoxyuridine (BrdU) (Abcam) (1:100), and rabbit monoclonal anti-ATM (Abcam) (1:30). The following secondary antibodies from Invitrogen were used for immunofluorescence at the dilutions specified: donkey anti-rabbit Alexa Fluor 594 IgG (1:250), donkey anti-mouse Alexa Fluor 488 IgG (1:250), donkey anti-goat Alexa Fluor 594 IgG (1:250), and goat anti-rat fluorescein isothiocyanate (FITC) from Southern Biotechnology (1:70).
Immunostained cells were scored for specific phenotypes by observation with a Nikon Eclipse E-400 epifluorescence microscope using a 100× oil immersion objective. Confocal images were captured with a confocal laser scanning microscope (Olympus FV500) using a 100× oil immersion objective and Fluoview software. Sequential scans of the Alexa 488 and Alexa 594 channels were performed to prevent bleed through between the channels. Composite images were assembled using Adobe Photoshop CS5 software.
Western blot analysis.Cells cultured on 35-mm-diameter plates were either left uninfected or infected with Ad5 or E4-. At the desired time, cells were washed twice with ice-cold PBS and scraped into a microcentrifuge tube and pelleted by centrifugation. The supernatant was aspirated, and the cell pellet was suspended in lysis buffer (Cell Signaling) (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). The extracts were then sonicated and centrifuged at top speed in a microcentrifuge for 1 min to remove insoluble material. The protein concentration of the extracts was quantified using the Bradford assay with Coomassie Plus (Pierce) according to the manufacturer's instructions and analyzed with a Nano-Drop ND-1000 spectrophotometer. Samples containing equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4% stacking and 5 to 10% resolving polyacrylamide gels depending upon the size of the protein. The separated proteins were then transferred to an enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham) overnight at 20 V and 4°C. The membranes were blocked with nonfat dry milk in TBST for 2 h at room temperature and incubated with specific primary antibody in TBST with 5% bovine serum albumin with shaking overnight at 4°C. Primary antibodies and the dilutions used for Western blotting were mouse monoclonal anti-phospho-ATM (p-S1981) (Abcam) (1:1,000), rabbit polyclonal anti-penton (a gift from U. Pettersson) (1:1,000), rabbit monoclonal anti-ATM (Abcam) (1:3,000), rabbit polyclonal anti-ATM (Novus) (1:1,000), and goat polyclonal anti-ATR (Santa Cruz) (1:200). The membranes were then subjected to multiple washes with TBST, after which they were incubated with the appropriate secondary antibody: either horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Amersham) (1:1,500) or alkaline phosphatase-conjugated anti-rabbit (Santa Cruz) (1:1,500). Secondary antibodies were diluted in 5% nonfat dry milk for 1 h at room temperature with shaking. After multiple washes, the membranes were incubated with ECL reagent (Amersham) to generate chemiluminescence signals, which were subsequently captured on ECL hyperfilm (Amersham). For protein quantification, alkaline phosphatase-conjugated secondary antibodies were used with enhanced chemifluorescence (ECF) substrate (Amersham) and analyzed with a STORM 860 phosphorimager (Molecular Dynamics). ImageQuant 5.2 (Molecular Dynamics) software was used to quantify the amount of protein.
Viral DNA analysis.Viral DNA was prepared as previously described (21). Briefly, cells grown on 35-mm-diameter plates were either left uninfected or infected with E4- or Ad5. Cells were lysed at 24 h postinfection (hpi) in proteinase K buffer (0.05 M Tris [pH 7.8], 0.0025 M EDTA, 0.25% SDS) and incubated with 0.4 mg/ml proteinase K for 4 h at 37°C, followed by phenol:chloroform extraction and ethanol-salt precipitation of nucleic acids. Total nucleic acid was subjected to RNase A (Fisher) digestion in Buffer H (Promega) followed by phenol:chloroform extraction and ethanol-salt precipitation. Dot blotting was performed using the RNA-free DNA. Samples containing 1.0 to 0.1 μg of DNA were denatured by boiling at 100°C for 10 min and immediately cooled on ice. Samples were adjusted to 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The samples were blotted on Hybond-N nylon membrane (Amersham) that was presoaked with 6× SSC using a dot blot manifold and applying vacuum pressure. The DNA was cross-linked to the membrane using a UV transilluminator. The membranes were then probed with Ad-specific radiolabeled probes generated using a Megaprime DNA labeling system (GE Healthcare) following the manufacturer's instructions. The hybridization reaction was performed with 5 × 106 cpm/ml probe at 65°C overnight. The amount of labeled DNA bound was detected using a STORM 860 phosphorimager (Molecular Dynamics) and quantified using ImageQuant 5.2 (Molecular Dynamics) software.
RNAi analysis.Cells were grown on 35-mm-diameter tissue culture dishes to 50% confluence and then transfected with ON-Targetplus small interfering RNA (siRNA) for target proteins ATM and ATR (Dharmacon Technologies) or a nontargeting siRNA (negative control) at 25 pmol/plate with 2 μl of lipofection reagent according to the manufacturer's instructions. Knockdown of target protein expression was analyzed at 72 and 96 h posttransfection (hpt) by Western blotting. Cells transfected with the respective siRNAs were infected with viruses at 72 hpt, and the samples were prepared for analysis at 24 h postinfection (hpi).
Pulsed-field gel electrophoresis (PFGE).Total cellular DNA was prepared as described previously (13). Briefly, cells were grown in 35-mm-diameter plates and were either left uninfected or infected with E4- and Ad5 viruses and at 24 hpi were treated with trypsin to remove them from the plate and embedded in low-melting-point agarose plugs. The agarose-embedded cells were subjected to proteinase K digestion overnight at 50°C. The plugs were then incubated with 50 mM EDTA for 8 h and loaded on 1.2% agarose gels for electrophoresis using a CHEF-DR II pulsed-field electrophoresis system (Bio-Rad) in hexagonal mode for 10 h at 6 V/cm with a switch time of 15 s. Gels were stained with ethidium bromide and photographed using a UV photodocumentation system to visualize viral DNA concatemers.
DSBR kinase inhibitors.Caffeine (Sigma) was dissolved in water to make a stock solution of 100 mM and added 2 hpi to a final concentration of 10 mM until the desired time. KU60019 (Tocris) was dissolved in dimethyl sulfoxide (DMSO) to make a 10 mM stock solution and added to the culture at 2 hpi to a final concentration of 5 μM for the desired time.
BrdU labeling.Bromodeoxyuridine (BrdU) labeling was done as described previously (22). Briefly, cells were grown on cover glasses and infected with E4- or Ad5. BrdU was dissolved in medium to a final concentration of 150 mM and added to the cells at the desired time for 1 h of incubation. Cells were then fixed with paraformaldehyde as described above. Prior to immunostaining, the cells were treated with 4 N HCl for 30 min to denature the DNA, washed with PBS twice for 5 min each time, neutralized with 0.1 M sodium borate for 5 min, and washed twice for 5 min each time with PBS.
RESULTS
ATM and ATR are not required for E4 mutant genome concatenation.E4 mutant infection results in genome concatenation by NHEJ. We have previously found that caffeine, which inhibits both ATM and ATR, also inhibited E4 mutant genome concatenation (16), suggesting a possible role for these kinases. We performed siRNA knockdown experiments to assess the role of ATM and ATR in E4 mutant genome concatenation. HeLa cells were transfected with siRNAs targeted against ATM (siATM) or ATR (siATR) and cultured for 72 or 96 h. The level of ATM and ATR proteins was substantially reduced at both 72 and 96 hpt compared to the level seen with untransfected or control siRNA-treated (siC) cells (Fig. 1A). To determine the effect of ATM or ATR knockdown on E4 mutant genome concatenation, HeLa cells were transfected with siATM, siATR, or siC for 72 h and subsequently left uninfected or infected with Ad5 or E4- at a multiplicity of infection (MOI) of 30 FFU/cell. Samples were prepared at 24 hpi for PFGE to measure genome concatenation. Ad5 infection showed no genome concatenation under either set of treatment conditions as expected since Ad5 prevents activation of DNA repair pathways. Concatemers of multiple genome lengths were observed following E4- infection of siATM-, siATR-, or siC-transfected cells (Fig. 1B). These results indicate that neither ATM nor ATR is individually critical for inducing viral genome concatenation and confirm previous observations (10). We next wished to address the possibility that ATM and ATR might provide redundant functions in promoting E4 mutant genome concatenation. We performed siRNA knockdown of ATR in ATM− cells to assess the role of the combined lack of ATM and ATR on genome concatenation. ATM− cells are derived from a patient with ataxia telangiectasia (A-T). They carry a mutation in the ATM gene and fail to produce the ATM protein (23). After siATR transfection, ATM− cells showed a marked reduction of ATR compared to siC-treated cells at 72 and 96 hpt (Fig. 1C, left panels). ATM− cells transfected with siC or siATR for 72 h were either left uninfected or infected with E4- or Ad5 and assayed for genome concatenation by PFGE at 24 hpi (Fig. 1C, right panel). E4 mutant genome concatemers were still observed in both siC- and siATR-treated cells. Similar results were seen at 32 hpi (data not shown). Taken together, these results indicate that ATM and ATR are not required either individually or together for E4 mutant genome concatenation.
ATM and ATR are not required for genome concatenation. HeLa cells were either untreated (UT), transfected with control siRNAs (siC), or specific siRNAs targeting either ATM (siATM) or ATR (siATR) mRNAs. (A) Transfected cells were processed for Western blotting to determine the levels of ATM or ATR at 72 and 96 hpt, or analyzed for the presence of β-actin as a control. (B) Cells transfected with the indicated siRNAs were either left uninfected (UI) or infected for 24 h with Ad5 or E4- at 30 FFU/cell from 72–96 hpt, and total DNA was analyzed by PFGE to measure the level of viral DNA concatemers. (C) siRNA knockdown of ATR was performed in ATM− cells. ATM− cells were transfected with control or ATR specific siRNA and the levels of ATR and β-actin proteins were determined by Western blotting (left panel). Total DNA from transfected cells infected with Ad5 or E4- was analyzed by PFGE to measure the level of viral DNA concatemers (right panel) as described for B.
ATM inhibits E4 mutant late gene expression and DNA replication.E4 mutant phenotypes include defects in late gene expression and DNA replication, which are more severe at low MOI (19, 24, 25). The MRN complex is an important mediator of E4 mutant DNA replication and late gene expression phenotypes (2–5). MRN could regulate E4 mutant phenotypes directly or by its activation of the ATM and/or ATR kinases. We performed siRNA knockdowns of ATM and ATR to assess a possible role for these downstream kinases. HeLa cells were transfected with siATR or siATM for 72 h and then either left uninfected or infected with Ad5 or E4- at an MOI of 30 FFU/cell. At 24 hpi the cells were harvested and total cell lysates were analyzed by Western blotting using antiserum that detects the viral late protein penton, and the results are presented in Fig. 2. In control siRNA-treated cells, E4- showed a substantial defect in penton expression compared to Ad5, as expected. Transfection with siATR had little effect on penton levels in cells infected with either Ad5 or E4-, suggesting that ATR is not involved in this phenotype. In contrast, siATM-transfected HeLa cells showed a 3-fold increase in E4- penton levels compared with siC-transfected controls, suggesting that lack of ATM significantly rescues E4- late gene expression. Similar results were seen using an antibody against the viral late protein hexon (data not shown). Taken together, these results identify a role for ATM, but not ATR, in interfering with E4 mutant late gene expression.
ATM interferes with E4 mutant late protein production while ATR does not. HeLa cells were either transfected with control siRNAs (siC), or specific siRNAs targeting either ATM (siATM) or ATR (siATR) mRNAs. Cells were then either left uninfected (UI) or infected with Ad5 or E4- as described for Fig. 1, and at 24 hpi analyzed for the presence of the late protein penton by Western blotting. Representative Western blots of penton levels achieved in cells treated with siATM (A) or siATR (B) are shown in the top panels. Levels of the control protein β-actin are also shown. E4- penton protein levels were quantified by phosphorimaging analysis of Western blots from 3 independent experiments and expressed as the fraction of the level achieved by Ad5, which was set at 1 (A and B, bottom graphs). Error bars show the standard error of the mean. A one-tailed Student's t test analysis was performed on the data used to generate the graphs. Statistically significant (P < 0.05) differences between the columns are indicated. N.S. = not statistically significantly different.
We performed infections in ATM− cells, and in isogenic ATM+ cells complemented with a copy of the ATM gene, to further assess the role of the ATM kinase in E4 mutant phenotypes. ATM+ and ATM− cells were infected with Ad5 or E4- at an MOI of 30 FFU/cell. Whole-cell lysates were prepared at 24 hpi, and Western blot assays were performed using antiserum that detects the viral late protein penton. Late proteins were quantified by phosphorimaging, and the results are presented in Fig. 3A. E4- penton levels were about 3-fold higher in ATM− cells than they were in ATM+ cells. The results seen with ATM− cells are similar to the results we obtained via siRNA-mediated knockdown of ATM in HeLa cells (Fig. 2) and confirm that the presence of ATM interferes with E4 mutant late gene expression.
ATM interferes with E4 mutant DNA replication. ATM− and isogenic ATM+ cells complemented for ATM, were infected with Ad5 or E4- at an MOI of 30 FFU/cell unless otherwise indicated. At 24 hpi cells were harvested and processed for Western blot analysis to study viral late protein levels, or total DNA was prepared and analyzed by dot blotting to quantify viral DNA levels. (A) Top panel shows a representative Western blot using an antibody that detects the viral late protein penton. Levels of the control protein β-actin are also shown. E4- penton protein levels were quantified by phosphorimaging analysis of Western blots from 3 independent experiments and expressed as the fraction of the level achieved by Ad5, which was set at 1 (bottom graph). Error bars show the standard error of the mean. Statistically significant differences between the columns are indicated, as determined by one-tailed Student's t test. (B) Graphs showing the results of dot blot quantitation of viral DNA levels in experiments performed at 30 (top graph) and 3 (bottom graph) FFU/cell. E4- DNA levels were quantified from 3 independent experiments and expressed as the fraction of the level achieved by Ad5, which was set at 1. Error bars show the standard error of the mean. Statistically significant differences between the columns are indicated, as determined by one-tailed Student's t test.
Ad DNA replication defines the onset of the late phase and is required for the switch from early to late gene expression (26). We next measured levels of viral DNA in ATM+ and ATM− cells infected with Ad5 and E4- to determine if DNA replication was impacted by the presence or absence of ATM. The E4 mutant demonstrated a 3-fold increase in DNA in ATM− compared to ATM+ cells (Fig. 3B, top graph). These data indicate that the presence of ATM inhibits E4 mutant DNA replication. Furthermore, the 3-fold increase in E4 mutant DNA levels in ATM− cells (Fig. 3B, top graph) is sufficient to account for the 3-fold increase in E4 mutant late protein levels observed in ATM− cells relative to ATM+ cells (Fig. 3A, bottom graph), suggesting that the primary effect of ATM is inhibition of E4 mutant DNA replication.
The DNA replication defect of E4 mutants is more severe at low MOI (24, 25). We further assessed the ability of ATM− cells to support E4 mutant DNA replication by assessing viral DNA levels following infections at a lower MOI. ATM− and ATM+ cells were uninfected or infected with Ad5 or E4- at an MOI of 3 FFU/cell. Total DNA was prepared at 24 hpi and analyzed by dot blotting. The results are shown in Fig. 3B (bottom graph). E4- DNA levels increased about 5-fold in ATM− relative to ATM+ cells when infections were done at the lower MOI. E4- DNA levels were only about 2-fold reduced relative to Ad5 DNA levels in ATM− cells, indicating that the DNA replication defect of the mutant was substantially rescued in the absence of ATM.
The kinase activity of ATM is important for inhibiting E4 mutant DNA replication.The kinase activity of ATM is critical for generating the ATM-mediated signaling cascade and can be inhibited by caffeine or the more specific ATM inhibitor KU60019 (27). We performed experiments in the presence and absence of these drugs to assess the importance of ATM kinase activity for inhibiting E4 mutant DNA replication. HeLa cells were infected with Ad5 or E4- at an MOI of 3 FFU/cell, and at 2 hpi cells were either left untreated or treated with medium containing 10 mM caffeine or 5 μM KU60019. Cells were harvested at 24 hpi, and dot blotting was performed to measure viral DNA levels. The results are presented in Fig. 4. Caffeine and KU60019 treatment of HeLa cells increased E4- DNA levels by 10-fold and 30-fold, respectively, compared to the levels seen with control cells that did not receive the inhibitor. The increased magnitude of the effect of KU60019 treatment likely reflects the observation that in these experiments, E4- DNA levels were more severely reduced relative to Ad5 DNA levels in untreated cells than was the case with the untreated controls in the caffeine experiments. Variation in the DNA replication defect of E4 mutants has been reported previously (19). In the presence of both inhibitors, E4 mutant DNA levels rose to about one-third of Ad5 levels by 24 hpi. By 32 hpi, E4- DNA levels were very close to Ad5 levels in both the caffeine- and KU60019-treated infected cells (data not shown). Similar increases in E4- DNA levels were also observed in infected cells treated with the ATM inhibitor KU55933 (data not shown). When we used KU60019 to treat ATM− cells infected with E4- and Ad5, we saw very little effect on E4- DNA levels, suggesting that the observed increases in E4- DNA levels in KU60019-treated HeLa cells depend on the presence of ATM (data not shown). Taken together, these results indicate that the kinase activity of ATM is important for ATM-mediated inhibition of E4 mutant DNA replication.
The kinase activity of ATM is important for inhibiting E4 mutant DNA replication. HeLa cells were infected with Ad5 or E4- at an MOI of 3 FFU/cell and subsequently incubated with the ATM kinase inhibitors caffeine (A) or KU60019 (B) from 2–24 hpi. Control infected cells were incubated in medium without the inhibitors but containing the same amount of DMSO solvent used for the drug treatment where indicated. Total DNA samples from 3 independent experiments were analyzed by dot blotting to measure viral DNA levels. E4- DNA levels were expressed as the fraction of the level achieved by Ad5, which was set at 1. Error bars show the standard error of the mean. Statistically significant differences between the columns are indicated, as determined by one-tailed Student's t test.
In E4 mutant infections, pATM localizes at viral replication centers that contain the viral DNA binding protein E2-72kDa (9) and to foci that colocalize with input viral genomes (15). We performed immunofluorescence experiments with pATM antibody to determine if its localization to either of these sites was correlated with inhibition of DNA replication. HeLa cells were infected with Ad5 or E4- at an MOI of 3 FFU/cell, fixed at 5, 7, 12, and 17 hpi, and immunostained with antibodies against pATM or E2-72kDa. Immunofluorescence micrographs of the staining patterns are presented in Fig. 5. At 5 hpi, focal accumulation of pATM was observed in cells infected with both Ad5 and E4- (Fig. 5A). These cells were not yet expressing significant levels of viral early proteins as evidenced by the lack of detectable E2-72kDa staining. Cells with pATM foci were equally prevalent at 5 hpi with either Ad5 or E4- and were about six times more prevalent than the background level seen in the uninfected cell population (Fig. 5B). At 7 hpi with Ad5, we detected pATM in prominent foci in cells that were not yet expressing E2-72kDa (Fig. 5C, panels a to c) but pATM staining was either not detectable or much weaker in cells that had begun to express E2-72kDa and sometimes contained small E2-72kDa foci (Fig. 5C, panels d to f). This suggests that ATM activation is not sustained in Ad5 infections. At 7 hpi with E4-, pATM was still detected in foci in cells with primarily diffusely localized E2-72kDa (Fig. 5C, panels g to i). In cells that contained early replication centers, pATM and E2-72kDa were nearly always colocalized or closely juxtaposed (Fig. 5C, panels j to l). At 12 hpi, we could detect larger, more developed DNA replication centers in Ad5 infections. In these cells, pATM staining was usually not detected (Fig. 5C, panels m to o), although some cells contained residual pATM staining that localized to the periphery of the E2-72kDa centers (data not shown). At 12 hpi with E4-, E2-72kDa foci showed little increase in size compared to 7 hpi and were again nearly always observed colocalized or tightly juxtaposed to a pATM focus (Fig. 5C, panels p to r). At 17 hpi, Ad5 replication centers were larger and in most of the infected cells very little pATM was present (Fig. 5C, panels s to u). At 17 hpi with E4-, E2-72kDa foci had still not increased much in size and remained strongly colocalized with pATM (Fig. 5C, panels v to x).
Time course showing pATM localization and BrdU incorporation relative to DNA replication centers in Ad5 and E4- infections. HeLa cells were uninfected (UI) or infected with Ad5 or E4- at 3 FFU/cell for the times indicated and then fixed and immunostained with antibodies against pATM (phosphoepitope S1981), BrdU, or the viral DNA binding protein E2-72kDa (72K) to detect viral DNA replication centers. (A) Immunofluorescence confocal micrographs showing pATM (green) and E2-72kDa (red) staining, or phase contrast images of uninfected cells or cells infected with Ad5 and E4- at 5 hpi. (B) Uninfected cells and cells infected with Ad5 or E4- were blindly scored for the presence of pATM foci at 5 hpi, and the results presented as a graph showing the percentage of cells in the culture with pATM foci. (C) Immunofluorescence confocal micrographs showing pATM (green), E2-72kDa (red), and merged staining patterns observed at 7, 12 and 17 hpi with Ad5 or E4-. (D) Ad5 or E4- infected HeLa cells were incubated with 150 mM BrdU for 1 h at either 12 or 17 hpi. Immunofluorescence confocal micrographs showing BrdU (green), E2-72kDa (red), and merged staining patterns are shown. Cells with early-, intermediate-, or late-stage replication centers were identified based on the size and shape of the E2-72kDa foci. Similarly sized replication centers in Ad5 and E4- infections are marked with white arrowheads to facilitate comparison of BrdU incorporation at these centers. Cells with intermediate- and late-stage DNA replication centers were the most prevalent phenotype in Ad5 infections while cells with early stage replication foci were the most prevalent phenotype in E4- infections at these time points. Cells marked with an asterix are examples of cells with widespread incorporation of BrdU in cellular DNA.
Since E4 mutant E2-72kDa foci did not increase much in size during the time course, we were interested in determining if DNA synthesis was still occurring in those replication foci that had developed in the presence of pATM. We addressed this by comparing BrdU incorporation in Ad5- and E4 mutant-infected cells. HeLa cells infected with Ad5 or E4- were incubated with 150 mM BrdU for 1 h starting at 12 or 17 hpi. The cells were then fixed, denatured, and immunostained with antibodies detecting BrdU or E2-72kDa, and the results are presented in Fig. 5D. These experiments were complicated by strong labeling of cellular DNA in S-phase cells (see Fig. 5D cells marked with an asterisk), which made it difficult to determine the level of BrdU incorporation at E2-72kDa foci in these cells. We therefore focused on cells that had E2-72kDa foci but lacked widespread BrdU incorporation and were likely to be cells that had initiated viral DNA synthesis outside the S phase. Ad5-infected cells with early E2-72kDa foci showed detectable BrdU incorporation at the foci (Fig. 5D, panels a to c). Ad5-infected cells with intermediate-stage (Fig. 5D, panels g to i and p to r) and late-stage (Fig. 5D, panels m to o and v to x) replication centers labeled strongly with BrdU, indicating that they are very active for DNA synthesis. Cells infected with E4- also showed detectable BrdU incorporation at many of the early (Fig. 5D, panels d to f) and intermediate (Fig. 5D, panels j to l and s to u) E2-72kDa foci, indicating that DNA synthesis was not completely blocked at E4- replication centers. However, we did notice that incorporation of BrdU at E4 mutant E2-72kDa foci was often reduced compared to similarly sized Ad5 E2-72kDa foci (Fig. 5D; compare Ad5 and E4- DNA replication foci marked with arrowheads).
We also studied the effect of inhibiting ATM kinase activity with KU60019 on the development of E2-72kDa containing replication centers in time course experiments. Early replication foci were detected in relatively few of the 7 hpi cells (1% or less), but they initiated with roughly similar frequencies in Ad5 and E4- infections in the absence of KU60019 treatment (Fig. 6A). KU60019 treatment significantly reduced the level of pATM associated with E4- DNA replication centers, which were dramatically increased in size (Fig. 6B, top panel). KU60019 treatment also significantly reduced the level of pATM detected in cells infected with E4- by Western blotting (Fig. 6B, bottom panel). A prominent pATM band was detected in extracts from untreated cells infected with E4-. This band was absent in extracts prepared from KU60019-treated E4- infections and from Ad5-infected and uninfected cells regardless of KU60019 treatment. Cells infected with Ad5 and E4- were then scored for the presence of small early E2-72kDa foci and large replication centers in cultures that were either left untreated or treated with KU60019 from 2 to 12 or 2 to 17 hpi. The results are presented in Fig. 6C. At 12 hpi in the absence of inhibitor, about 50% of cells infected with both Ad5 and E4- contained focal concentrations of E2-72kDa. However, in E4- infections most of the E2-72kDa was in small early replication foci, while in Ad5 infections the majority of the cells contained larger intermediate- and late-stage replication centers. This indicates that in Ad5 infections, the small replication foci are transient and rapidly progress to larger replication centers. By 17 hpi in the absence of the ATM kinase inhibitor, 70% to 85% of both Ad5- and E4 mutant-infected cells had focal concentrations of E2-72kDa, and again, nearly all of the cells infected with E4- contained small early replication foci, while nearly all of the Ad5-infected cells contained larger intermediate- or late-stage replication centers. We found that KU60019 treatment allowed E4 mutant-infected cells to convert from early replication foci to large replication centers more efficiently. Large replication centers were present in about 10% of the cell population infected with E4- at 12 hpi and about 50% at 17 hpi (Fig. 6C). Inhibiting ATM kinase activity had little effect on the conversion of early replication foci to large replication centers in Ad5 infections. Taken together, these results indicate that although pATM does not prevent the formation of early replication foci in E4- infections, it substantially inhibits their development into larger DNA replication centers.
ATM inhibition facilitates progression of E4- DNA replication centers from small early replication foci to larger intermediate- and late-stage replication centers. HeLa cells were infected with Ad5 or E4- and then either incubated with medium containing DMSO only, or 5 μM KU60019 in DMSO to inhibit ATM kinase activity from 2 hpi until the times indicated. (A) The percentage of cells infected with Ad5 and E4- with E2-72kDa (72K) replication foci was scored at 7 hpi. (B) The top panel shows immunofluorescence micrographs of pATM and E2-72kDa staining at 17 hpi with Ad5 and E4- incubated in the presence or absence of KU60019. The bottom panel shows Western blot analysis of extracts prepared from uninfected (UI) cells or from cells infected with Ad5 or E4- at 17 hpi that were or were not treated with KU60019, and probed with antibodies against pATM, ATM, or β-actin. (C) The percentage of cells containing small (black bars) or large (gray bars) E2-72kDa containing DNA replication centers was determined in Ad5 and E4- infections that were and were not incubated with KU60019 from 2 hpi until the times indicated.
When we studied the localization of pATM relative to late-stage replication centers containing E2-72kDa at 17 hpi with wild-type Ad5, we observed that about 30% of the cells demonstrated unexpectedly high levels of staining with the pATM antibody (data not shown). In these cells, the pATM antibody staining was dispersed throughout the nucleus and did not colocalize with E2-72kDa. The widespread pATM antibody staining was still observed in Ad5-infected cells treated with KU60019, even though this drug effectively inhibited the appearance of pATM foci in cells infected with E4- (Fig. 6B, top panel). We also saw no evidence of pATM in Western blot analyses of extracts from Ad5 infections, although a clear band corresponding to pATM was seen in extracts from cells infected with E4- (Fig. 6B, bottom panel). These results suggest the possible presence of a cross-reacting epitope that is upregulated during the late phase of Ad5 infections. We did not see evidence of this cross-reacting epitope being upregulated in E4- infections, even when DNA replication was rescued by KU60019 treatment.
Mre11 and ATM localization to E4 mutant replication centers is not sufficient to prevent E4 mutant DNA replication when ATM kinase activity is inhibited.Mre11 and pATM localize to viral DNA replication centers in E4 mutant infections (9, 10), and Mre11 is known to bind E4 mutant DNA (5). Our previous results indicate that the interaction of MRN complex proteins with E4 mutant genomes is important for inhibiting DNA replication (5). We studied the localization of Mre11 at E4 mutant DNA replication centers following treatment with KU60019 to determine if inhibition of ATM kinase activity affected recruitment of Mre11 to replication centers. HeLa cells were infected with E4- at an MOI of 3 FFU/cell, and at 2 hpi, cells were either left untreated or incubated with medium containing 5 μM KU60019 until 17 hpi. Infected cells were then fixed and immunostained with antibodies against Mre11 and E2-72kDa. The results are presented in Fig. 7A. In E4- infections, Mre11 colocalized with most E2-72kDa centers in both the presence and absence of KU60019. E4 mutant replication centers were significantly larger in cells treated with KU60019. These results indicate that ATM kinase inhibition does not prevent the association of Mre11 with E4 mutant replication centers. We also studied the localization of Mre11 following E4- infection of ATM− cells at 3 FFU/cell. At 17 hpi, cells were fixed and immunostained with antibodies against Mre11 and E2-72kDa. Mre11 colocalized with E2-72kDa in ATM− cells (Fig. 7A). Taken together, these data indicate that neither ATM nor its kinase activity is required for Mre11 localization at E4 mutant DNA replication centers. Since E4- DNA replication is substantially rescued in KU60019-treated infections (Fig. 4 and 6) and in ATM− cells (Fig. 3), the results also suggest that localization of Mre11 at replication centers is not sufficient to inhibit E4- DNA replication in the absence of ATM or its kinase activity.
Localization of Mre11 or ATM to viral replication centers does not interfere with E4- DNA replication when the ATM kinase is inactivated. (A) HeLa or ATM− cells were infected with E4- at 3 FFU/cell and fixed for immunofluorescence at 17 hpi. The indicated infections were incubated with 5 μM KU60019 from 2–17 hpi to inhibit ATM kinase activity. Immunofluorescence micrographs showing E2-72kDa (green) and Mre11 (red) and merged staining patterns are shown. (B) Nbs1+, Nbs1−, and HeLa cells were infected with E4- at 3 FFU/cell and fixed for immunofluorescence at 17 hpi. E4- infected HeLa cells were incubated with 5 μM KU60019 from 2–17 hpi to inhibit ATM kinase activity. Immunofluorescence micrographs showing E2-72kDa (72K), pATM, ATM, and merged staining patterns are shown.
We next investigated pATM and ATM localization following E4- infection of Nbs1− cells that fail to make a functional MRN complex (18) and ATM localization in HeLa cells that were treated with KU60019 to inhibit ATM kinase activity. Cells were infected with E4- at an MOI of 3 FFU/cell, and at 17 hpi, the cells were fixed and immunostained with the indicated antibodies. The results are presented in Fig. 7B. We saw very little pATM in Nbs1− cells compared to Nbs1+ cells. E4 mutant replication foci were significantly larger in Nbs1− cells, which is consistent with previous observations that E4 mutant DNA replication is rescued in these cells (Fig. 7B, left panels) (5). Unphosphorylated ATM did not localize to E4- replication centers in Nbs1− cells, as expected, since Nbs1 is required to recruit ATM to sites of DNA damage (Fig. 7B, right panels) (18, 28). In HeLa cells treated with KU60019 to inhibit ATM kinase activity, unphosphorylated ATM was still recruited to viral DNA replication centers. Since KU60019 significantly rescues E4 mutant DNA replication (Fig. 4 and 6), these results indicate that ATM recruitment to viral replication centers does not itself interfere with viral DNA replication if the ATM kinase is inactive. Taken together, these results indicate that a functional MRN complex is needed for activation of pATM, and for the association of ATM with E4- DNA replication centers, but this is not sufficient to interfere with viral DNA replication if the ATM kinase is not active.
DISCUSSION
We have sought to clarify the role of the ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. Cells lacking either ATM or ATR still concatenate E4 mutant genomes (Fig. 1) (10). However, previous reports indicated that genome concatenation is sensitive to the ATM/ATR inhibitor caffeine (3, 16), suggesting a possible role for these kinases. We performed siRNA knockdown of ATR in ATM− cells to address the possibility that these kinases might functionally substitute for each other, and to our surprise, we found that E4 mutant genomes were still concatenated (Fig. 1). ATR knockdown effectively prevented Chk1 phosphorylation in E4- infections (data not shown), suggesting that knockdown was sufficient for at least this ATR-mediated activity. Although we cannot rule out the possibility that ATR knockdown was insufficient to prevent genome concatenation, we think it is likely that caffeine affects genome concatenation by a mechanism that does not involve ATM and ATR kinase inhibition. DNAPK exhibits sensitivity to caffeine in vitro (29), although DNAPK-dependent NHEJ in vivo is resistant (29, 30). Nevertheless, it is possible that high concentrations of caffeine or prolonged incubation times could affect DNAPK, which is a critical enzyme for E4 mutant genome concatenation (13). Alternatively, caffeine could affect an as-yet-unidentified substrate critical for genome concatenation.
The MRN complex inhibits E4 mutant DNA replication (2–5), but the mechanism involved is not understood. ATR does not inhibit E4 mutant late gene expression (Fig. 2) or DNA replication (3). In contrast, when E4 mutant infections were analyzed in cells that lack ATM, we saw similar increases in both viral DNA and late protein penton levels in experiments performed at an MOI of 30 FFU/cell (Fig. 2 and 3), suggesting that the primary effect of ATM is inhibition of viral DNA replication, which is required for late gene expression. Previous work (19) indicated that E4 mutants still display a measurable defect in late gene expression following high MOI infections that show a slight or no DNA replication defect. This may explain why E4- DNA levels were within 70% of Ad5 values in ATM− cells (Fig. 3B, top graph) whereas E4- penton levels still demonstrated a significant 3-fold reduction relative to Ad5 in ATM− cells (Fig. 3A). Our data suggest that the increase in E4- late gene expression in cells lacking ATM most likely reflects an increase in viral DNA levels that occurs in these cells, but ATM may not fully complement the component of the late gene expression defect that is independent of viral DNA replication. The E4- DNA replication phenotype was more severe when cells were infected at a lower MOI of 3 FFU/cell. We also observed a more dramatic rescue of E4- DNA levels in the absence of ATM (Fig. 3B, lower graph) and following treatment with ATM kinase inhibitors (Fig. 4). These results confirm the ability of ATM and its kinase activity to interfere with E4 mutant DNA replication and are in contrast to those of Lakdawala et al. (3), who reported that E4 mutant DNA replication was still severely defective in A-T cells that lack ATM. Possible differences between our experiments and theirs include the MOI and the source of the A-T cells used. Although we do not know the reason for the discrepancy between our results and those of Lakdawala et al. (3), we have observed an increase in E4 mutant DNA replication in ATM− cells compared with an isogenic ATM+ cell line complemented with ATM (Fig. 3B), in HeLa cells treated with ATM kinase inhibitors (Fig. 4), and following siRNA knockdown of ATM in HeLa cells (data not shown). Thus, multiple lines of evidence support our conclusion that ATM inhibits E4 mutant DNA replication, particularly following low-MOI infections. Interestingly, E4 mutant DNA replication was rescued to similar extents (about 50% of Ad5 levels) following low-MOI infection in ATM− cells (Fig. 3B, lower graph), in Nbs1− cells that lack a functional MRN complex (data not shown), and in siATM-treated HeLa cells (data not shown). This supports the idea that the ability of the MRN complex to inhibit E4 mutant DNA replication is likely due to its ability to activate ATM. This does not rule out ATM-independent roles for the MRN complex in other E4 mutant phenotypes. For example, ATM is not required for the ability of MRN to promote E4 mutant genome concatenation (Fig. 1).
Karen and Hearing have previously shown that pATM colocalizes with foci containing incoming viral DNA in E4 mutant infections (15), and pATM is associated with E4 mutant replication centers that contain the E2-72kDa DNA replication protein at later times (10). Localization of pATM with either incoming or replicating viral genomes could potentially interfere with E4 mutant DNA replication. Interestingly, we observed focal accumulation of pATM in Ad5-infected cells similar to the pATM foci seen in E4 mutant infections. Ad5 pATM foci were clearly visible at 5 hpi before a detectable level of the early protein E2-72kDa was expressed (Fig. 5A and B). However, once early proteins are detected, the intensity of pATM staining was greatly reduced in Ad5-infected cells (Fig. 5C, panels d to f). We think it is likely that either E4- or Ad5 infection can trigger focal concentration of pATM at locations containing input viral DNA at early times. Early gene expression, including that of the Ad5 E4 proteins, would then be expected to inactivate the MRN complex, and this likely interferes with sustained pATM activation in Ad5 infections. Although pATM was observed in early foci before the onset of DNA replication in E4- infections (Fig. 5B), this did not prevent the timely development of small replication foci containing the E2-72kDa DNA binding protein (Fig. 6A and C). In contrast, we observed a dramatic effect on the conversion of small replication foci to larger E2-72kDa-containing centers in E4- infections with active pATM (Fig. 5C and 6C). E4- replication centers also showed less intense incorporation of BrdU (Fig. 5D) compared with Ad5 results. ATM kinase inhibition did not affect the overall percentage of E4 mutant-infected cells with E2-72kDa foci, but it significantly increased the ability of the E4 mutant to convert small DNA replication foci into larger DNA replication centers (Fig. 6C). Our data suggest that pATM does not completely block E4 mutant DNA replication but significantly reduces its efficiency.
The ability of MRN to localize at viral DNA replication centers correlates with its ability to inhibit E4 mutant DNA replication (4). The Nbs1 C-terminal domain is important for binding Mre11 (28) and for inhibiting E4 mutant DNA replication (3). Furthermore, we have found that Nbs1 is important for the ability of Mre11 to both bind E4- DNA and inhibit DNA replication (5). These results are consistent with a model in which Nbs1-dependent recruitment of Mre11 to viral DNA is important for inhibiting E4 mutant DNA replication. Nevertheless, recruitment of Mre11 to viral DNA may not be sufficient for this effect. We found that Mre11 is still recruited to viral replication centers in cells that lack ATM and when the ATM kinase activity has been inhibited (Fig. 7A), and yet this is not sufficient to block E4 mutant DNA replication (Fig. 3 to 6). Lakdawala et al. (3) found that a C-terminal fragment of Nbs1 that binds Mre11 but fails to bind ATM was still able to recruit the MRN complex protein Rad50 to viral replication centers but was unable to interfere with E4 mutant DNA replication. Although not interpreted as such, these results are completely consistent with our observations that the MRN complex is needed to activate pATM, allow it to localize at E4 mutant DNA replication centers (Fig. 7B), and inhibit DNA replication (Fig. 3 to 6). Taken together, these results suggest a model in which Ad genomes are delivered to the nucleus and initially induce the focal accumulation of pATM at locations containing viral genomes (15) (Fig. 5A). We propose that inactivation of Mre11 by E4 proteins prevents sustained activation of pATM in Ad5 infections, which are subsequently able to efficiently develop large late-stage DNA replication centers (Fig. 5 and 6). In E4 mutant infections, pATM activation is sustained due to the failure to inactivate Mre11, and pATM remains tightly associated with early DNA replication foci and the larger intermediate-stage DNA replication centers that develop inefficiently in the presence of pATM (Fig. 5 and 6). Activated ATM could thus inhibit DNA replication when it is associated with centers containing viral DNA.
The mechanism by which ATM interferes with E4 mutant DNA replication is unknown. Repair of double-strand breaks following ionizing radiation involves extensive chromatin changes in the vicinity of the break. Many DDR proteins are recruited to the ends of the break and form foci that can be observed by microscopy; these include MRN complex proteins, pATM, which can phosphorylate the histone variant H2AX (γH2AX), Mdc1, 53BP1, and BRCA1 (31). If a large complex of proteins assembles at the ends of E4 mutant genomes during activation of DDRs, this could possibly interfere with the ability of the viral DNA replication proteins to identify the terminally located Ad origin of replication and initiate viral DNA synthesis (5, 15). In view of this model, it is interesting that in E4- infections, early replication foci initiated in cells with a frequency similar to that seen with Ad5 (Fig. 5C and 6A), although they failed to develop into the large late-stage replication centers typically seen in Ad5 infections (Fig. 5C and 6C). These results suggest that the ability of viral replication proteins to initiate E4 mutant DNA replication is not completely blocked by DDR activation but that E4 mutant DNA replication progresses inefficiently. Possible roles for activated ATM in interfering with E4 mutant DNA replication could include altering the chromatin configuration of viral DNA and thus making replication inefficient, phosphorylating specific substrates that interfere with viral DNA replication, or reducing DNA replication efficiency by affecting cell cycle checkpoints. Further experiments to elucidate these mechanisms are in progress and should help us to better understand how cellular DDRs interfere with E4 mutant DNA replication.
ACKNOWLEDGMENTS
We are very grateful to Ulf Pettersson, Tommy Linné, and Arnold Levine for providing antibodies and Pat Concannon for providing the Nbs1− and Nbs1+ cell lines used in this study. We also thank Joseph Carlin for critically reading the manuscript and all the members of our laboratory for their suggestions and support.
This research was supported by the National Cancer Institute (grant CA82111) and by awards from Miami University.
FOOTNOTES
- Received 5 February 2013.
- Accepted 24 May 2013.
- Accepted manuscript posted online 5 June 2013.
- Address correspondence to Eileen Bridge, BridgeE{at}MiamiOH.edu.
REFERENCES
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