WRNIP 1 protects stalled forks from degradation and promotes fork restart after replication stress
2016; Springer Nature; Volume: 35; Issue: 13 Linguagem: Inglês
10.15252/embj.201593265
ISSN1460-2075
AutoresGiuseppe Leuzzi, Veronica Marabitti, Pietro Pichierri, Annapaola Franchitto,
Tópico(s)Semiconductor materials and devices
ResumoArticle30 May 2016free access Source DataTransparent process WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress Giuseppe Leuzzi Giuseppe Leuzzi Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Veronica Marabitti Veronica Marabitti Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pietro Pichierri Pietro Pichierri Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Annapaola Franchitto Corresponding Author Annapaola Franchitto orcid.org/0000-0003-4232-4727 Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Giuseppe Leuzzi Giuseppe Leuzzi Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Veronica Marabitti Veronica Marabitti Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pietro Pichierri Pietro Pichierri Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Annapaola Franchitto Corresponding Author Annapaola Franchitto orcid.org/0000-0003-4232-4727 Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Author Information Giuseppe Leuzzi1, Veronica Marabitti1, Pietro Pichierri2 and Annapaola Franchitto 1 1Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy 2Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy *Corresponding author. Tel: +39 0649903042; Fax: +39 0649903650; E-mail: [email protected] The EMBO Journal (2016)35:1437-1451https://doi.org/10.15252/embj.201593265 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 Abstract Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA. We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability. Synopsis The hitherto poorly characterized ATPase WRNIP, originally identified as an interactor of the Werner syndrome helicase, is found to cooperate with the RAD51 recombinase in the protection of stalled replication forks. WRNIP1 protects stalled replication forks from degradation. The ATPase activity of WRNIP1 promotes fork restart after replication stress. WRNIP1 contributes to the stabilization of RAD51 on stalled replication forks. Downregulation of the anti-recombinase FBH1 compensates for loss of WRNIP1 activity. Introduction The proper execution of DNA replication is an essential aspect of cellular life. Proliferating cells are constantly subjected to a wide variety of threats originating by the action of exogenous and endogenous agents that can hinder replication fork progression. Several studies have demonstrated that inaccurate handling of stalled replication forks can lead to genomic instability, a well-known source of human diseases and cancer onset (Carr & Lambert, 2013; Magdalou et al, 2014). To minimize such a risk, cells have evolved sophisticated mechanisms to cope with perturbed replication forks (Branzei & Foiani, 2009, 2010; Yeeles et al, 2013; Zeman & Cimprich, 2014). The importance of stabilizing and restarting stalled replication forks is also evidenced by the increasing number of proteins identified as being part of these mechanisms. Accordingly, multiple pathways work in the recovery of replication stalling, and some homologous recombination (HR) proteins have been implicated in preserving the integrity of arrested replication forks (Petermann & Helleday, 2010; Costanzo, 2011). Indeed, a current model proposes that BRCA2 and RAD51 may act in preventing rather than repairing lesions at stalled replication forks, to protect nascent DNA strand from degradation mediated by the exonucleolytic activity of MRE11 (Hashimoto et al, 2010; Schlacher et al, 2011; Ying et al, 2012). Among proteins participating in the maintenance of genome stability, whose function is still poorly characterized, is the human Werner helicase interacting protein 1 (WRNIP1). WRNIP1 was identified as a binding partner of the Werner protein (WRN) (Kawabe Kawabe et al, 2001, 2006), a member of the RecQ family of DNA helicases that plays a crucial role in response to replication stress, and significantly contributes to the recovery of stalled replication forks (Rossi et al, 2010; Franchitto & Pichierri, 2014). WRNIP1 belongs to the AAA+ class of ATPase family proteins that is evolutionary conserved (Kawabe Kawabe et al, 2001; Hishida et al, 2001). Although the yeast homolog of WRNIP1, MGS1, is required to prevent genome instability caused by replication arrest (Branzei et al, 2002), little is known about the function of human WRNIP1. However, in vitro studies support the possibility that the ATPase activity of WRNIP1 could stimulate DNA polymerase delta (Polδ) to re-initiate DNA synthesis, for example after fork arrest, through a physical association with WRN and Polδ (Tsurimoto et al, 2005). Further in vitro investigations reveal that WRNIP1 binds in an ATP-dependent manner to forked DNA that mimics stalled replication forks (Yoshimura et al, 2009). Furthermore, WRNIP1 foci overlap with replication factories, reinforcing the hypothesis of its function at replication forks (Crosetto et al, 2008). In this study, we have identified an uncharacterized function of WRNIP1 at perturbed replication forks. Loss of WRNIP1 results in DNA damage accumulation due to the inability of cells to properly protect stalled replication forks from nucleolytic attack by MRE11. We demonstrate that WRNIP1 is recruited to stalled replication forks. We further show that WRNIP1 interacts with the BRCA2/RAD51 complex and contributes to the stabilization of RAD51 on ssDNA to preserve stalled fork integrity. Interestingly, we prove that blocking the removal of RAD51 from chromatin by depleting FBH1, the MRE11-mediated degradation of stalled replication forks as well as chromosomal aberrations are counteracted in WRNIP1-deficient cells. Furthermore, we establish that WRNIP1 is implicated in the stalled fork resumption through its ATPase activity. Altogether, our work suggests a molecular basis for the role of human WRNIP1 in safeguarding genome stability in response to replication stress. Results WRNIP1 is required for protection and restart of stalled forks upon replication stress To investigate the function of human WRNIP1 during DNA replication, we monitored replication perturbation genomewide at single-molecule level by performing DNA fibre assay. Firstly, we generated MRC5SV cells stably expressing WRNIP1-targeting shRNA (shWRNIP1). Next, isogenic cell lines stably expressing the RNAi-resistant full-length wild-type WRNIP1 (shWRNIP1WT) or its ATPase-dead mutant form of WRNIP1 (shWRNIP1T294A) (Tsurimoto et al, 2005) were created using the shWRNIP1 cells (Fig 1A). To determine whether WRNIP1 affects replication under normal growth conditions (i.e. in the absence of any treatment), we measured the rate and symmetry of the replication fork progression in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. We sequentially labelled cells with the thymidine analogues 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU) as described in the experimental scheme (Fig 1B). Under these conditions, shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells showed almost identical fork velocity with an average fork progression rate of about 1.0 kb per minute (Fig 1C). Moreover, the frequency of asymmetric replication tracks was similar in all cell lines (Fig 1D), confirming that no elongation defect is triggered when WRNIP1 or its enzymatic activity was lost. Figure 1. Loss of WRNIP1 leads to nascent DNA strand degradation after HU-induced replication stress Western blot analysis showing the expression of the WRNIP1 protein in wild-type cells (shWRNIP1WT) and WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells. MRC5SV fibroblasts were used as a positive control. The membrane was probed with an anti-FLAG or anti-WRNIP1. GAPDH was used as a loading control. Below each lane of the blot the ratio of WRNIP1 protein to total protein, then normalized to MRC5SV, is reported. Experimental scheme of dual labelling of DNA fibres in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU and then subjected to a pulse-labelling with IdU. Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks was measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t-test). Cells were treated as in (B). For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. A schematic representation of symmetric and asymmetric forks is given. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (that is, outside the violet dashed lines in the graphs), the fork was considered asymmetric. The percentage of asymmetric forks was calculated for all cell lines. N = number of forks counted for each cell line. R represents linear correlation coefficient. Experimental scheme of dual labelling of DNA fibres in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, treated with 4 mM HU and then subjected to a pulse-labelling with IdU. Graphs show the percentage of red (CldU) tracts (stalled forks) or red-green (CldU-IdU) contiguous tracts (restarting forks) in the cells. Means are shown, n = 3. Error bars represent standard error (*P < 0.05; **P < 0.01; Student's t-test). Representative DNA fibre images are shown. Scale bar, 10 μm. Experimental scheme of dual labelling of DNA fibres in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were sequentially pulse-labelled with CldU and IdU as indicated, then treated or not with 4 mM HU. Representative IdU tract length distributions in all cell lines under unperturbed conditions (top graph) or after HU treatment (bottom graph). Median tract lengths are given in parentheses. See also Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are shown. Scale bar, 10 μm. Source data are available online for this figure. Source Data for Figure 1 [embj201593265-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint To obtain a deeper insight into the role of WRNIP1 in replication, we explored whether loss of WRNIP1 influences fork progression after HU-induced replication stress. Thus, we pulse-labelled shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells with CldU and IdU as reported (Fig 1E). DNA fibre analysis showed that WRNIP1 depletion resulted in a significant enhancement in the percentage of stalled forks induced by HU with respect to wild-type cells (Fig 1F). Similarly, the expression of the mutant form of WRNIP1 greatly affected fork progression after HU (Fig 1F). Interestingly, comparing the percentage of restarting forks in all cell lines, we observed that loss of WRNIP1 reduced the ability of cells to resume replication after release from HU in the same extent as loss of its ATPase activity (Fig 1F). All other replication parameters were not significantly different among the cell lines (Appendix Fig S1A and B). These results implicate WRNIP1, through its ATPase activity, in restarting stalled forks. We next verified whether WRNIP1 was involved in the protection of stalled forks, by examining the stability of nascent replication strands. To this aim, we changed the DNA labelling scheme. Thus, shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were sequentially pulse-labelled with CldU and IdU to mark nascent replication tracts before fork stalling with HU (Fig 1G). The maintenance of the IdU label after HU treatment measures the extent of fork stability on the stretched DNA fibres. The analysis showed that IdU tract length remained unchanged with or without HU treatment in cells expressing wild-type WRNIP1 (shWRNIP1WT) (7.72 and 7.96 μm, respectively; Fig 1H). On the contrary, in WRNIP1-deficient cells (shWRNIP1), fork stalling led to a significant shortening of IdU tract length compared to unperturbed replication (4.70 and 7.43 μm, respectively; Fig 1H). Notably, in shWRNIP1T294A cells, IdU tract length was left unaffected after HU as in wild-type cells, revealing that the ATPase activity is dispensable for protection of stalled forks (7.30 and 7.40 μm, with and without HU, respectively; Fig 1H). Since nascent IdU tracts are formed before treatment with HU, it is plausible that the decrease in length of the IdU tracts takes place during exposure to the drug, as previously demonstrated (Schlacher et al, 2011). Thus, we deduced that WRNIP1 is essential in avoiding degradation of nascent DNA strands at stalled forks. To determine whether the phenotype of WRNIP1-deficient cells is a general response to replication arrest, we pulse-labelled shWRNIP1WT and shWRNIP1 cells with IdU, followed by exposure to a high dose of aphidicolin (Aph), a selective inhibitor of the replicative DNA polymerases (Appendix Fig S2A). Since we observed that Aph showed substantial similarity to HU in the ability to reduce IdU tract length in the absence of WRNIP1 (7.34 and 4.83 μm, shWRNIP1WT and shWRNIP1, respectively; Appendix Fig S2B), we concluded that replication stress caused by various agents needs WRNIP1 to protect stalled forks. Moreover, to ascertain whether the role of WRNIP1 is kept in other cell types, we tested HEK293T cells transfected with control siRNA (HEK293TsiCtrl) or WRNIP1 siRNA (HEK293TsiWRNIP1). After transfection, cells were pulse-labelled with IdU and then exposed to HU (Appendix Fig S3A). Although similar IdU tract length was observed in both cell lines under unperturbed conditions, however, WRNIP1-deficient cells (HEK293TsiWRNIP1) exhibited a defective maintenance of nascent length tracts after HU treatment as compared to the wild-type cells (HEK293TsiCtrl) (4.42 and 7.34 μm, respectively; Appendix Fig S3B). This confirms that the fork-protective role of WRNIP1 is independent from the cell lines. Overall, our results suggest that, when replication is perturbed, WRNIP1 maintains the integrity of stalled forks and ensures their restart via its ATPase activity. MRE11 nuclease activity is responsible for degradation of nascent DNA strand at stalled forks in the absence of WRNIP1 It has been reported that MRE11 activity is responsible for degradation of HU-stalled forks in BRCA2-defective cells (Schlacher et al, 2011; Ying et al, 2012). Since we proved that WRNIP1-deficient cells show instability of stalled forks, which is reminiscent of that observed in the absence of BRCA2, we asked whether MRE11 nuclease could similarly promote fork degradation in our cells. To test this hypothesis, we double-labelled shWRNIP1WT and shWRNIP1 cells, followed by treatment with HU and mirin, a chemical inhibitor of MRE11 activity (Dupré et al, 2008); then, we measured the length of the IdU tracts (Fig 2A). As expected, mirin had no effect on HU-treated wild-type cells (Fig 2B). However, we found that loss of MRE11 activity prevented IdU tract shortening by HU treatment in the absence of WRNIP1, reaching a value comparable to that of wild-type cells (7.89 and 4.95 μm, with or without MRE11 inhibition, respectively; Fig 2B). Figure 2. Inhibition of MRE11 exonuclease activity prevents nascent DNA strand degradation after replication stress Experimental scheme of dual labelling of DNA fibres in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 μM mirin. Representative IdU tract length distributions in shWRNIP1WT (top graph) or shWRNIP1 cells (bottom graph) after treatment. Median tract lengths are given in parentheses. See Appendix Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fibre images are shown. Scale bar, 10 μm. Download figure Download PowerPoint Next, to exclude off-target effects produced by the MRE11 inhibitor, shWRNIP1 cells were transfected with siRNAs directed against MRE11, then labelled with IdU and treated with HU (Appendix Fig S4A). Depletion of MRE11 resulted in a clear evidence of protection from nascent strand degradation during HU exposure, as IdU tract length was longer in HU-treated cells in which MRE11 was abrogated (7.43 and 4.72 μm, with or without MRE11 knockdown, respectively; Appendix Fig S4B). Therefore, we conclude that MRE11 nuclease activity degrades stalled forks in the absence of WRNIP1. WRNIP1 depletion results in parental-strand ssDNA accumulation and RAD51 destabilization after fork stalling Next, we tested whether WRNIP1 depletion caused an increased parental-strand ssDNA accumulation at replication forks due to degradation of nascent DNA strand. We specifically visualized ssDNA by immunofluorescence using an anti-IdU antibody under non-denaturing conditions. To this aim, shWRNIP1WT and shWRNIP1 cells were labelled with IdU for 24 h and then released into fresh culture medium for 2 h before stalling forks with HU (Fig 3A). Moreover, to assess the dependence of ssDNA formed on MRE11 activity, parallel samples were exposed to mirin (Fig 3A). Our analysis showed that WRNIP1-deficient cells presented higher amount of ssDNA than wild-type cells under unperturbed and HU-treated conditions (Fig 3A). However, MRE11 inhibition substantially lowered the accumulation of ssDNA detected with or without fork stalling only in shWRNIP1 cells (Fig 3A). Experiments with HU-treated shWRNIP1WT and shWRNIP1 cells, in which MRE11 activity was disrupted by RNAi, confirmed the nuclease-dependent formation of ssDNA at parental strand in the absence of WRNIP1 (Appendix Fig S5). Then, to verify whether nascent strand became single-stranded at stalled forks, shWRNIP1WT and shWRNIP1 cells were shortly labelled with IdU immediately before HU treatment (Appendix Fig S6A). Immunofluorescence analysis showed little, but similar, IdU labelling in both shWRNIP1WT and shWRNIP1 cells after HU treatment (Appendix Fig S6). Figure 3. Analysis of parental ssDNA formation and RAD51 destabilization at stalled replication forks Evaluation of ssDNA accumulation at parental-strand by immunofluorescence analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity was chemically inhibited with 50 μM mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines and error bars represent the mean ± SE; n = 3 (ns, not significant; **P < 0.01; ****P < 0.0001; two-tailed Student's t-test). Representative images are shown. DNA was counterstained with DAPI (blue). Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. Lamin B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (input) in the cells was determined with the relevant antibodies. Lamin B1 was used as a loading control. In the graph, the fold increase with respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/total RAD51 (or MRE11) is reported for each cell line. Analysis of DNA–protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental design used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA and subjected to PLA assay as described in the Materials and Methods section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51, respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows data presented as mean ± SE of the number of PLA spots per cell from three independent experiments (ns, not significant; **P < 0.01; two-tailed Student's t-test); n = 3. Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. Lamin B1 and GAPDH were used as loading controls (input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitated DNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldU Co-IP proteins)/the total of labelled DNA immunoprecipitated with CldU (CldU-IP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2. Source data are available online for this figure. Source Data for Figure 3 [embj201593265-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Since RAD51-ssDNA complex is functionally relevant in protecting stalled replication forks from degradation (Schlacher et al, 2011), we wondered whether the greater amount of ssDNA detected in WRNIP1-deficient cells could correlate with a larger amount of RAD51 bound to chromatin. To address this point, we performed a Western blot analysis after cellular fractionation in shWRNIP1WT and shWRNIP1 cells treated or not with HU as indicated (Fig 3B). As shown in Fig 3B, the amount of chromatin-bound RAD51 was lower in shWRNIP1 than in shWRNIP1WT cells under both unperturbed and fork-stalling conditions. Furthermore, as expected, in wild-type cells we observed an enhanced chromatin loading of MRE11 after fork stalling (Mirzoeva & Petrini, 2003); however, in WRNIP1-deficient cells, we detected a greater increase (Fig 3B). In agreement with our biochemical fractionation experiments, immunofluorescence detection of RAD51 relocalization in shWRNIP1WT and shWRNIP1 cells, treated or not with HU, showed a reduced percentage of RAD51 foci in the absence of WRNIP1 after fork stalling (Appendix Fig S7). We further confirmed the presence of low levels of RAD51 in WRNIP1-deficient cells. Using a modification of the in situ proximity ligation assay (PLA), a fluorescence-based improved method that makes possible to reveal physical protein–protein interaction (Söderberg et al, 2008), to detect protein/DNA association (Iannascoli et al, 2015), we next investigated the co-localization of RAD51 at/near ssDNA. To this aim, shWRNIP1WT and shWRNIP1 cells were treated or not with HU (Fig 3C). We found that the co-localization between ssDNA (anti-IdU signal) and RAD51 significantly decreased in shWRNIP1 cells after replication stress (Fig 3C). Since high amount of ssDNA formation was revealed in shWRNIP1 cells (Fig 3A), and given that visualization of a red spot in the cell requires the presence of both ssDNA (anti-IdU signal) and RAD51, the smaller number of PLA spots observed in the absence of WRNIP1 may correlate with the reduced levels of RAD51. Finally, to exclude the possibility that, in shWRNIP1 cells, RAD51 was susceptible to proteasome-mediated degradation, we examined the amount of RAD51 upon MG132 treatment alone or in combination with HU. We found that proteasomal inhibition led to accumulation of RAD51 in unperturbed shWRNIP1 cells, but not after fork stalling (Appendix Fig S8). Therefore, we concluded that, under replication stress, RAD51 is not degraded but likely not properly stabilized in the absence of WRNIP1. Altogether these findings indicate that, when cells are depleted for WRNIP1, fork stalling results in a large enhancement of ssDNA at template DNA strand produced by the action of MRE11 nuclease activity, which does not lead to a greater amount of RAD51 bound to chromatin. RAD51 and MRE11 are differently recruited to stalled replication forks in WRNIP1-deficient cells Our experiments suggest that loss of WRNIP1 results in reduced RAD51 loading to chromatin and MRE11-dependent nascent strand degradation after fork stalling. Thus, we wanted to ascertain whether RAD51 and MRE11 were differently recruited to stalled replication forks in the absence of WRNIP1. To this end, shWRNIP1WT and shWRNIP1 cells were pulse-labelled with CldU to mark newly replicated DNA and exposed or not to HU. Co-immunoprecipitation of RAD51 or MRE11 with CldU-labelled replication sites was performed from cross-linked chromatin to detect DNA-associated proteins at replication forks. Equal loading of proteins was evaluated by Ponceau S staining, and equal amounts of immunoprecipitated DNA from each sample were verified by dot blot analysis (Fig 3D). In line with previous studies (Petermann et al, 2010; Somyajit et al, 2015), our CldU-IP experiments confirmed the loading of RAD51 at nascent strand in HU-treated wild-type cells (Fig 3D). Interestingly, although in shWRNIP1 cells, RAD51 was present at sites of stalled replication forks, the level was significantly lower than that in wild-type cells (Fig 3D). On the contrary, the amount of MRE11 was higher in the absence of WRNIP1 as compared to wild-type cells (Fig 3D). Moreover, and in accordance with a previous study (Dungrawala & Cortez, 2015), our experiments indicated that WRNIP1 co-immunoprecipitated with CldU-labelled replication sites after HU treatment in wild-type cells, proving that WRNIP1 is associated with stalled replication forks (Fig 3D). Consistently with the MRE11-mediated nascent strand degradation, these results provide evidence for enhanced recruitment of MRE11, but reduced level of RAD51 at stalled replication forks in WRNIP1-deficient cells. RAD51 protects nascent DNA strand from degradation after replication stalling in WRNIP1-deficient cells The RAD51 recombinase is directly implicated in the protection of nascent strand from MRE11-mediated degradation (Hashimoto et al, 2010; Schlacher et al, 2011), and BRCA2 stimulates RAD51 assembly on ssDNA (Jensen et al, 2010; Liu et al, 2010; Moynahan & Jasin, 2010). Since loss of WRNIP1 leads to a phenotype similar to that observed in BRCA2-defective cells, to identify the pathway in which WRNIP1 functions under replication stress, we examined whether chemical inhibition of RAD51, which disrupts RAD51 binding to DNA (Huang & Mazina, 2012), could affect stabilization of stalled forks in WRNIP1-deficient cells. To this end, shWRNIP1WT and shWRNIP1 cells were exposed to IdU and RAD51 inhibitor and treated with HU; then, the length of the IdU tracts was measured (Fig 4A). As expected, HU treatment resulted in IdU tract shortening in WRNIP1-deficient cells, but not in wild-type cells (5.31 and 7.40 μm, respectively; Fig 4B). Moreover, as previously demonstrated (Schlacher et al, 201
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