ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery
2010; Springer Nature; Volume: 29; Issue: 18 Linguagem: Inglês
10.1038/emboj.2010.205
ISSN1460-2075
AutoresFrancesca Ammazzalorso, L.M. Pirzio, Margherita Bignami, Annapaola Franchitto, Pietro Pichierri,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle27 August 2010free access ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery Francesca Ammazzalorso Francesca Ammazzalorso Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Livia Maria Pirzio Livia Maria Pirzio Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Margherita Bignami Margherita Bignami Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Annapaola Franchitto Corresponding Author Annapaola Franchitto Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pietro Pichierri Corresponding Author Pietro Pichierri Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Francesca Ammazzalorso Francesca Ammazzalorso Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Livia Maria Pirzio Livia Maria Pirzio Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Margherita Bignami Margherita Bignami Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Annapaola Franchitto Corresponding Author Annapaola Franchitto Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pietro Pichierri Corresponding Author Pietro Pichierri Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Author Information Francesca Ammazzalorso1, Livia Maria Pirzio1, Margherita Bignami1, Annapaola Franchitto 1 and Pietro Pichierri 1 1Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy *Corresponding authors. Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. Tel.: +39 064 990 3042; Fax: +39 064 990 3650; E-mail: [email protected] or Tel.: +39 064 990 2994; Fax: +39 064 990 3650; E-mail: [email protected] The EMBO Journal (2010)29:3156-3169https://doi.org/10.1038/emboj.2010.205 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM-dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C-terminal S/TQ residues. Suppression of ATR-mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co-localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM-unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest. Introduction An accurate response to stressed replication is crucial for maintaining genome stability. If perturbed replication forks are not properly handled, cells may accumulate chromosomal rearrangements as frequently observed in cancers and a subset of genetic diseases collectively referred to as chromosome fragility syndromes. The Werner syndrome (WS) is a chromosome fragility and cancer-prone disease caused by mutations in the human RecQ helicase WRN (Muftuoglu et al, 2008). Several pieces of evidence suggest that the primary function of WRN is related to recovery of stalled replication forks. At the biochemical level, WRN shows a remarkable preference towards substrates that mimic structures associated with stalled replication forks (Brosh et al, 2002; Choudhary et al, 2004; Machwe et al, 2006) and, at the cellular level, loss of WRN causes S-phase defects, hypersensitivity to agents interfering with replication and fragility of chromosomal regions that are considered natural hotspots of replication fork stalling (Poot et al, 1999, 2001; Pichierri et al, 2001; Pirzio et al, 2008; Sidorova et al, 2008). Consistently, replication fork arrest triggers extensive subnuclear re-localisation of WRN and co-localisation with RPA and replication foci (Constantinou et al, 2000; Rodriguez-Lopez et al, 2003; Franchitto and Pichierri, 2004). The exact contribution of WRN to replication fork recovery is not fully appreciated, but it has been suggested that it may facilitate replication restart by either promoting recombination or by processing intermediates at stalled forks in a way that counteracts unscheduled recombination (Pichierri, 2007; Sidorova, 2008). Recent data from our group indicate that loss of WRN determines activation of an alternative pathway of fork recovery resulting in DSB accumulation that is subsequently repaired through recombination (Franchitto et al, 2008), supporting the hypothesis that, upon replication fork stalling, WRN acts to limit fork collapse. Recovery of stalled replication forks involves the co-ordinated action of several proteins under the control of the replication checkpoint kinase ATR (Cimprich and Cortez, 2008). Loss of ATR function determines hypersensitivity to replication fork arrest and accumulation of chromosomal breakage in response to stressed replication (Cliby et al, 1998; Casper et al, 2002; Cortez, 2003), which are phenotypes reminiscent of those associated with WS cells. Indeed, WRN is phosphorylated after replication arrest in an ATR-dependent manner and both ATR and WRN act in a common pathway to stabilise common fragile sites (Pichierri et al, 2003; Pirzio et al, 2008). However, it is currently unknown if WRN is a substrate of ATR and which are the functional consequences of this phosphorylation on the recovery of stalled forks. We examined the phosphorylation of WRN by ATR and evidenced that WRN can be phosphorylated at multiple sites located at its C-terminal region. We found that phosphorylation of WRN is functionally related to the prevention of fork collapse and DSB accumulation carried out by ATR upon replication arrest. Interestingly, we observed that ATR and ATM-dependent phosphorylation of WRN influence different steps of the replication fork recovery reaction. Although phosphorylation by ATR regulates WRN subnuclear re-localisation and interaction with RPA, preventing DSB formation at stalled forks, ATM-dependent phosphorylation becomes essential during recovery of collapsed forks influencing the ability of RAD51 to form nuclear foci. Results ATR phosphorylates the WRN protein at multiple sites on its C-terminal region The minimal consensus sequence of ATR or ATM kinase is the Ser/Thr-Gln (S/TQ) motif and the human WRN protein contains six S/TQ motifs clustered at the C-terminal region, suggesting that it could be a direct substrate of ATR and/or ATM kinase activity. As ATR, but not ATM, is the primary kinase involved in the response to replication stress, we first evaluated whether the WRN S/TQ sites were phosphorylated by ATR. To this aim, HeLa cells, in which ATR was depleted (Figure 1A), were treated with campthotecin (CPT) or hydroxyurea (HU) to induce replication arrest and cell lysates were subjected to WRN immunoprecipitation. Using an antibody that specifically recognises phospho-S/TQ motifs (pS/TQ), we found that phosphorylation of WRN was barely detectable in untreated conditions and increased greatly after treatments (Figure 1B). Interestingly, depletion of ATR resulted in reduced WRN immunoreactivity to the anti-pS/TQ antibody, suggesting that most, if not all, of the phosphorylation is ATR related under perturbed replication (Figure 1B). To analyse which of the six S/TQ sites of WRN (S991, S1058, S1141, T1152, S1256 and S1292) were the candidate targets of ATR, we mutated several S/TQ sites in WRN simultaneously (Figure 1C). To exclude any phosphorylation by a co-precipitating kinase, we first verified by ATR immunocomplex kinase assay that the wild-type (wt) fragment of WRN was efficiently phosphorylated by ATR wt, but not by the kinase-dead form of the enzyme (ATRkd) (Figure 1C). Similarly, the analysis using the M1 fragment containing the two Ala substitutions at previously identified ATM substrates (Kim et al, 1999; Matsuoka et al, 2007) as well as the M2, in which two additional residues were mutated, were efficiently phosphorylated by ATR (Figure 1C), clearly suggesting that ATR and ATM do not target the same residues. However, phosphorimaging analysis showed that the M3 fragment still presented a residual level of phosphorylation (about 32% of wt, Figure 1C), whereas the M4, containing all the six potential S/TQ sites changed into Ala, was not phosphorylated at all (Figure 1C), excluding the presence of non-canonical ATR phosphorylation sites. To identify the actual ATR phosphorylation sites, we generated the M5 fragment in which S991, T1152 and S1256 were mutated into Ala and the M6 fragment containing Ala substitutions at the ATM putative substrates (Figure 1C). The M5 fragment was not phosphorylated by ATR, whereas M6 showed a level of phosphorylation similar to that of the wt fragment (Figure 1C). To confirm the observed non-overlapping phosphorylation by ATR and ATM, we performed ATM kinase assays using the M5 (ATRdead) and M6 (ATMdead) fragments as substrates. The results presented in Figure 1D confirmed that the S/TQ residues phosphorylated by ATR are not substrate for ATM and vice versa. Interestingly, addition of the ATM inhibitor KU55933 to the assays prevented phosphorylation of the M5 fragment, excluding phosphorylation by other kinases (Figure 1D). Figure 1.WRN is phosphorylated by ATR at multiple sites of the C-terminal region. (A) Depletion of ATR. HeLa cells were transfected with control siRNAs or siRNAs directed against ATR, and 48 h later, cell lysates were subjected to immunoblotting with anti-ATR antibody. Tubulin was used as loading control. (B) Evaluation of WRN phosphorylation in ATR-depleted cells. After siRNA transfection, HeLa cells were treated with 2 mM HU or 10 μM CPT for 6 h. Cell extracts were immunoprecipitated (IP) using anti-WRN antibody followed by immunoblotting with an anti-pST/Q antibody. Total WRN was used to evaluate the amount of WRN immunoprecipitated. (C) Identification of ATR phosphorylation sites on the C-terminal region of WRN by immunocomplex kinase assay. Top, schematic representation of the GST-tagged C-terminal wild-type or mutant forms of WRN. Locations of multiple Ala substitutions are indicated. Below, in vitro kinase assay. GST-C-terminal wild-type (wt) or mutants WRN (M1, M2, M3, M4, M5 and M6) were incubated with the wild-type or inactive form of ATR immunopurified from HeLa cells in the presence of 32P-ATP (for details see ‘Materials and methods’). After separation by SDS–PAGE, the presence of phosphorylation was assessed by phosphorimaging. The amount of C-terminal WRN fragments used is shown by Coomassie staining. Phosphorylation levels were expressed as the percentage of residual phosphorylation of each mutant fragment compared with the wild type. (D) Analysis of in vitro phosphorylation of C-terminal WRN fragment by ATM. The wild type (wt) and both the M5 and M6 mutant fragments containing, respectively, Ala changes at the ATR or at the ATM phosphorylation sites were incubated with immunopurified Flag-ATM with or without 10 μM KU55933 (iATM). (E) Analysis of mutant WRN phosphorylation. HEK293T cells were transfected with plasmids expressing a Flag-tagged full-length WRN wild type or carrying Ala substitutions at all the six S/TQ sites (6A) and 48 h later treated with 2 mM HU or 10 μM CPT for 6 h. Cell extracts were immunoprecipitated (IP) using anti-WRN antibody following immunoblotting with an anti-pST/Q antibody. Anti-Flag tag antibody was used to evaluate the amount of wild-type or mutant form WRN immunoprecipitated. Download figure Download PowerPoint Altogether, in vitro kinase assays indicate that S991, T1152 and S1256 are ATR substrates, suggesting that the C-terminal fragment of the protein can undergo multiple phosphorylation events also in vivo. To confirm that the C-terminal WRN region might be phosphorylated in multiple residues, we transiently expressed in HEK293T cells an N-terminal-truncated fragment of WRN (WRNΔN) and analysed the presence of phosphorylated species of the fragment. The immunoblotting analyses revealed that Myc-WRNΔN migrated as a doublet consisting of two species, designed as β and γ, in both untreated and treated extracts (Supplementary Figure 1). Treatment of extracts from either control or CPT-treated cells with λ-phosphatase eliminated both the β and γ forms of Myc-WRNΔN and resulted in the appearance of a single faster-migrating species that we named α form, confirming that the β and γ forms represented phosphorylated species (Supplementary Figure 1). To exclude that WRN could be phosphorylated at additional S/TQ sites after replication arrest, we transfected HEK293T cells with constructs expressing the wt full-length Flag-tagged WRN protein (FlagWRNwt) or a mutant in which all the six C-terminal S/TQ sites were changed into Ala (Flag-WRN6A). Immunoblotting analysis of cells immunoprecipitated using anti-Flag antibody showed that, with or without treatment, mutation of the six C-terminal S/TQ sites completely abrogated WRN phosphorylation, as assessed by anti-pS/TQ antibody (Figure 1E). Altogether, these results show that WRN is an ATR substrate both in vitro and in vivo, evidencing that multiple C-terminal phosphorylation sites can be targeted in response to replication arrest. Efficient re-localisation of WRN in nuclear foci and co-localisation with RPA require ATR phosphorylation Having shown that ATR and ATM do not target the same WRN residues and that in response to perturbed replication WRN is phosphorylated by ATR, the WRN6A protein can be considered as an ATR-unphosphorylable mutant under these experimental conditions. To assess whether phosphorylation by ATR could influence the nuclear dynamics of WRN after replication stress, we generated cell lines stably expressing a Flag-tagged wt WRN (WSWRN) or the WRN6A mutant (WSWRN6A). Even though WSWRN6A cells expressed levels of the WRN protein similar to that of WS cells (Figure 2A), hypersensitivity to replication stressing agents was comparable with that seen in WS cells (Figure 2B). Moreover, immunofluorescence analysis showed that formation of WRN foci in WSWRN6A cells was greatly reduced after HU-induced replication fork stalling as compared with cells expressing wt WRN (Figure 2C). Consistently with previous reported data (Constantinou et al, 2000), in WSWRN cells, HU treatment led to re-localisation of WRN in nuclear foci co-localising with RPA at sites of replication fork stalling in the majority of the nuclei (Figure 2D). In contrast, only a minor fraction of the nuclei positive for WRN foci revealed co-localisation with RPA in WSWRN6A cells (Figure 2D). In addition, nuclear foci formed by the mutant form of WRN preferentially co-localised with the DSB-marker γ-H2AX (Supplementary Figure 2), suggesting that they might pinpoint spontaneous DSBs or collapsed replication forks. Interestingly, cells expressing the WRN3A mutant, containing Ala substitutions only at the identified ATR phosphorylation sites (S991, T1152 and S1256; WSWRN3A), showed a defect in WRN subnuclear re-localisation and co-localisation with RPA similar to that observed in the WRN6A mutant (Figure 2C and D). Figure 2.WRN re-localisation and co-localisation with RPA upon replication arrest depend on its phosphorylation by ATR. (A) Western blotting on extracts from WS cells stably expressing the Flag-tagged wild-type WRN (WSWRN), the 6A mutant (WSWRN6A) or the ATR-unphosphorylable form of WRN (WSWRN3A) showing levels of WRN using an anti-WRN antibody. WS cells were used as negative control and tubulin as loading control. (B) Cells were treated with different doses of HU or CPT for 16 h and allowed to grow in drug-free medium for 2 weeks before analysis of clonogenic survival. Survival is expressed as percentage of the untreated cultures. Data are presented as mean±s.e. from three independent experiments. (C) Analysis of WRN re-localisation to nuclear foci after replication arrest. Images show WRN nuclear distribution with or without 8 h treatment. The graph shows the percentage of WRN-positive nuclei. Data are presented as means of three independent experiments. Error bars represent standard errors. (D) Analysis of WRN and RPA co-localisation after replication arrest. Cells were treated with 2 mM HU for 8 h and subjected to immunofluorescence using anti-WRN and anti-RPA32 antibodies. Representative images from cells treated with HU for 8 h are presented. Insets show an enlarged portion of the nuclei for a better evaluation of the co-localisation status of WRN with RPA32 foci. Scale bars, 10 μm. Download figure Download PowerPoint These studies indicate that phosphorylation by ATR is essential for correct WRN subnuclear re-localisation at sites of replication fork stalling after HU-induced replication stress. ATR phosphorylation of WRN prevents the appearance of DSBs upon replication fork stalling In the absence of WRN, stalled forks undergo collapse and DSBs are formed (Franchitto et al, 2008). To assess whether ATR phosphorylation was required for the protective function of WRN at stalled forks, WS, WSWRN, WSWRN6A and the WSWRN3A cells were analysed for their ability to accumulate DSBs. In WS cells, HU-induced replication fork stalling resulted in a large number of nuclei with high γ-H2AX fluorescence (Figure 3A), which appeared reduced in percentage and intensity by expressing the wt form of WRN (Figure 3A). In contrast, expression of either the completely unphosphorylable form of WRN (WRN6A) or the ATR-unphosphorylable mutant (WRN3A) induced an accumulation of γ-H2AX-positive nuclei as the absence of WRN (Figure 3A). Noteworthy, expression of the WRN6A mutant led to enhanced number of γ-H2AX-positive nuclei with highly fluorescence respect to that observed in parental WS cells (Figure 3A). Figure 3.WRN phosphorylation by ATR is required to prevent accumulation of DNA breakage after replication arrest. (A) Analysis of DNA breakage using γ-H2AX immunostaining. Cells were exposed to HU for the indicated times and stained with an antibody against γ-H2AX. In the panel, representative images from each experimental point are shown. Graphs show the percentage of γ-H2AX-positive nuclei with medium or high intensity of γ-H2AX fluorescence. (B) DNA breakage as detected using a neutral comet assay. Cells were treated with HU for the indicated times and then subjected to comet assay. In the panel, representative images are shown. Data are presented as mean tail moment and as means of three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. (C) Depletion of ATR. WS, WSWRN or WSWRN6A cells were transfected with control siRNAs or siRNAs directed against ATR and cell lysates subjected to immunoblotting with anti-ATR antibody. Tubulin was used as loading control. (D) Analysis of DNA breakage by γ-H2AX in WS, WSWRN or WSWRN6A cells in which ATR was depleted using RNAi. After transfection, cells were treated with 2 mM HU for the indicated times and stained with an antibody against γ-H2AX. In the panel, representative images are shown. Graphs show the percentage of γ-H2AX-positive nuclei. Data are presented as means of three independent experiments and error bars represent standard errors. Scale bars, 10 μm. Download figure Download PowerPoint As γ-H2AX immunostaining does not necessarily mark only DSBs upon replication arrest, we used neutral comet assay to verify accumulation of these lesions at stalled forks in the WRN phosphorylation mutants. Comet assay confirmed that HU treatment determined the formation of DSBs in WS cells, which could be prevented by expressing the wt form of WRN (Figure 3B) and showed that WSWRN6A cells, consistently with the γ-H2AX data, showed levels of breakage even more abundant than those observed in the absence of WRN or in the presence of the WRN3A mutant (Figure 3B). It has been previously reported that ATR functions in preserving fork stability after replication stress (Cortez, 2003; Chanoux et al, 2009). As we observed that the expression of the unphosphorylable mutant form of WRN leads to accumulation of DSBs after replication arrest, we asked whether ATR-mediated avoidance of DSBs at stalled forks might directly involve phosphorylation of WRN. Thus, we depleted ATR function by RNAi in WSWRN, WS and WSWRN6A cells (Figure 3C) to analyse the level of DSB accumulation after HU treatment. As shown in Figure 3D, abrogation of ATR in wt cells (WSWRN+siATR) treated with HU led to a two-fold increase in the percentage of nuclei with γ-H2AX foci respect to the WSWRN cells. However, the absence of ATR in WRN-deficient (WS+siATR) cells or expressing the unphosphorylable form of WRN (WSWRN6A+siATR) did not enhance the effect of HU-induced accumulation of DSBs (Figure 3D). Similar results were obtained from comet assay (data not shown). To confirm that ATR phosphorylation of WRN is specifically related to the function of ATR in the stability of stalled forks, we analysed activation of the S-M checkpoint in cells expressing the WRN6A mutant. Progression into mitosis in the presence of HU was impaired only in ATR RNAi-treated cells (Supplementary Figure 3), suggesting that the function of ATR in promoting cell cycle arrest is unrelated to phosphorylation of WRN. Our data are consistent with the hypothesis that phosphorylation of WRN may mediate the ATR-dependent function of replication fork stability preservation, avoiding degeneration of forks into DSBs. WRN phosphorylation is required for the recovery from perturbed replication Given the function of WRN phosphorylation in the correct handling of stalled forks and the observation that expression of WRN6A leads to a higher sensitivity to HU than the absence of the WRN protein, we evaluated the possibility that loss of S/TQ phosphorylation of WRN could affect the ability of the cells to recover cell cycle progression after replication stress. Flow cytometry analyses showed that WSWRN cells readily resumed cell cycle progression after release from the HU-induced block and completely recovered from the arrest within 18–24 h (Figure 4A). Similarly, WS cells arrested in response to HU exposure recovered normally cell cycle progression, even if with a delayed kinetic (24 h) if compared with the wt counterpart (Figure 4A). In contrast, at 12 h from release, the majority of WSWRN6A cells were still accumulated in S phase (Figure 4A), and even though they restarted cell cycle progression at later times, they also underwent extensive cell death (Figure 4A and B). Figure 4.WRN phosphorylation is necessary for the cellular recovery from prolonged replication arrest and for replication fork restart. (A) Analysis of cell cycle progression after replication arrest. Cells were treated overnight with 2 mM HU, samples collected at the indicated recovery times and subjected to FACS analysis. (B) Evaluation of cell viability by LIVE/DEAD assay. WS, WSWRN or WSWRN6A cells were treated with 2 mM HU for the indicated times. Cell viability was evaluated as described in ‘Materials and methods’. Data are presented as per cent of dead cells and as means of three independent experiments. Error bars represent standard errors. (C) Schematic representation of experimental design used to measure replication fork recovery and examples of replication track labelling after recovery, labelled replication tracks showing stalled forks recovering upon HU removal (a) or forks collapsed after HU treatment (b). (D) Graph shows quantification of restarting forks evaluated by dividing the number of restarting forks (i.e. ‘a’ tracks) by the total number of forks (i.e. a+b). WS, WSWRN, WSWRN6A or WSWRN3A cells were treated with 2 mM HU for the indicated times and recovered for 1 h in IdU-containing medium before DNA fibre assay. Data are presented as means±s.e. from three independent experiments. *Statistically significant (P<0.01) by a Student's t-test. (E) Images of DNA fibres visualised by immunofluorescence detection. Images derived from samples treated with HU for 16 h. Scale bars, 20 μm. Download figure Download PowerPoint To test whether cell death observed in WSWRN6A cells depended on defective restart of stalled/collapsed forks after DSB formation, we analysed the ability of stalled forks to recover DNA synthesis by the DNA fibre assay as previously described (Merrick et al, 2004; Franchitto et al, 2008). Using this protocol, stalled forks, which resume DNA synthesis once HU is removed, show both IdU and CldU labelling, whereas forks that have been inactivated present only IdU labelling (Figure 4C). After release from 8 h HU treatment, almost the totality of stalled forks recovered in WSWRN cells, whereas WS and WSWRN6A cells showed a reduced number of restarting forks, as shown by poor CldU labelling (Figure 4D). However, and in contrast with WS cells, prolonged replication arrest (16 h) resulted in a further reduction of restarting forks in WSWRN6A cells (Figure 4D and E). On the other hand, expression of WRN3A, which has mutated only the ATR phosphorylation sites, did not result in a defect in fork restart greater than that observed in the absence of WRN (Figure 4D and E). Taken together, these results indicate that expression of the ATR and ATM-unphosphorylable WRN6A mutant leads to a more severe phenotype than that observed in WS cells or in cells expressing the WRN3A mutant, especially after DSB formation and fork collapse, affecting the ability of cells to resume replication under replication stress condition. Loss of phosphorylation of WRN affects the RAD51-dependent recovery of stalled forks The reduced ability of WSWRN6A cells to resume from S-phase arrest suggests that the completely unphosphorylable WRN protein may act in a dominant-negative manner, affecting the other pathways involved in the recovery from replication blockage in the absence of an active WRN. As WS cells use RAD51-dependent recombination to recover from replication arrest (Franchitto et al, 2008), we investigated whether loss of ATR and ATM phosphorylation of WRN could interfere with engagement of recombination. In WS cells, increased RAD51 foci formation correlates with enhancement of recombination (Franchitto et al, 2008), thus, we used RAD51 foci as readout of its involvement in replication recovery. As expected, in WS cells, HU treatment increased formation of RAD51 foci, which is reverted by expressing wt WRN (Figure 5A). However, WSWRN6A cells showed levels of RAD51-positive nuclei comparable with those observed in WSWRN cells (Figure 5A), but the amount of DSBs was equivalent to that exhibited by the parental WS cells (Figure 5B). Furthermore, treatment with etoposide, an agent inducing DSBs irrespective of DNA replication, resulted in similar percentages of RAD51-positive nuclei in all the cell lines, suggesting that defective accumulation of RAD51 foci seen in WSWRN6A cells was specific for DSBs created at collapsed forks (Supplementary Figure 4). Then, we determined whether the elevated HU sensitivity of the WSWRN6A cells could be attributable to concomitant impairment of WRN- and RAD51-dependent replication fork recovery mechanisms. Given that viability of WS cells after prolonged replication arrest is affected b
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