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Checkpoint-Dependent and Independent Roles of the Werner 12628–12639 Nucleic Acids Research, 2014, Vol. 42, No. 20 Published online 28 October 2014 doi: 10.1093/nar/gku1022 Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress Giorgia Basile1,2,†, Giuseppe Leuzzi1,2,†, Pietro Pichierri2,3 and Annapaola Franchitto1,2,* 1Section of Molecular Epidemiology, Department of Environment and Primary Prevention, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy, 2Genome Stability Group, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy and 3Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanita,` Viale Regina Elena, 299-00161 Rome, Italy Received July 22, 2014; Revised October 08, 2014; Accepted October 09, 2014 ABSTRACT agents perturbing DNA replication (1–3). Based on WRN enzymatic activities and substrate preferences in vitro,it Werner syndrome (WS) is a human chromosomal in- is thought that WRN may participate in multiple DNA stability disorder associated with cancer predispo- metabolic pathways in vivo, such as replication, recombi- sition and caused by mutations in the WRN gene. nation and repair (4–6). WRN has been implicated in the WRN helicase activity is crucial in limiting breakage correct and fruitful recovery from replication fork arrest at common fragile sites (CFS), which are the prefer- (1,7–8). Given a coordinate action of WRN and DNA poly- ential targets of genome instability in precancerous merase delta in the replication of DNA substrates contain- lesions. However, the precise function of WRN in re- ing G4 tetraplex structures (9), the crucial requirement of sponse to mild replication stress, like that commonly the WRN helicase activity in maintaining common frag- used to induce breaks at CFS, is still missing. Here, ile site (CFS) stability and a role of WRN in alleviating we establish that WRN plays a role in mediating CHK1 stalled forks arrested at secondary structures formed at CFS FRA16D (10,11), WRN could work in the removal of the activation under moderate replication stress. We pro- obstacles encountered by the replisome during replication vide evidence that phosphorylation of CHK1 relies on to facilitate fork progression. the ATR-mediated phosphorylation of WRN, but not Several studies suggested a possible cross-talk between on WRN helicase activity. Analysis of replication fork WRN and ATR (12–14). ATR deficiency is associated with dynamics shows that loss of WRN checkpoint me- CFS expression (15), and defects of other checkpoint pro- diator function as well as of WRN helicase activity teins, including CHK1, lead to instability at CFS (16). Of hamper replication fork progression, and lead to new interest, we previously demonstrated that WRN regulates origin activation to allow recovery from replication CFS stability by acting in a pathway associated with ATR slowing upon replication stress. Furthermore, by- (10). Despite WS cells exhibit an ATR-like instability at pass of WRN checkpoint mediator function through CFS (10), and that WRN has been found phosphorylated overexpression of a phospho-mimic form of CHK1 by ATR under replication stress (12), there is no evidence of a functional requirement of WRN in the establishment of restores fork progression and chromosome stability the replication checkpoint upon moderate replication per- to the wild-type levels. Together, these findings are turbation such as the one that causes breaks at CFS. the first demonstration that WRN regulates the ATR- In this study, we disclose a previous unidentified role for checkpoint activation upon mild replication stress, WRN in mediating the ATR-checkpoint activation under preventing chromosome fragility. mild replication stress. Moreover, our findings provide a novel mechanistic framework to the WRN-dependent sta- bilization of CFS. INTRODUCTION The Werner syndrome protein, WRN, is a member of the RecQ family of DNA helicases mutated in the cancer-prone disease Werner syndrome (WS). WS cells show a marked delay in S-phase progression and are extremely sensitive to *To whom correspondence should be addressed. Tel: +39 0649903042; Fax: +39 0649903650; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2014, Vol. 42, No. 20 12629 MATERIALS AND METHODS chromosome preparations and fluorescence in situ hy- bridization (FISH) analysis was performed and analyzed as Cell cultures previously reported (10). HEK293T cells were obtained from American Type Cul- ture Collection (VA, USA). HEK293T cells proficient and DNA fiber analysis deficient for WRN were generated by stably expressing, Cells were pulse-labeled with 25 ␮M chlorodeoxyuridine respectively, a scrambled shRNA (shCTRL) or shRNA (CldU) and then labeled with 250 ␮M iododeoxyuridine against WRN (shWRN) (OriGene). Cells were cultured (IdU) at the times specified, with or without treatment as in the presence of hygromycin (200 ␮g/ml; EMD Chemi- reported in the experimental schemes. DNA fibers were cals Inc.) to maintain selective pressure for shRNA expres- prepared and spread out as previously described (12). For sion. AG11395 (WRN-deficient) fibroblasts retrovirally- immunodetection of labeled tracks, the following primary transduced with full-length cDNA encoding wild-type antibodies were used: rat anti-CldU/BrdU (Abcam) and WRN (WSWRN), missense-mutant form of WRN with in- mouse anti-IdU/BrdU (Becton Dickinson). Images were active helicase (WRN-K577M) or expressing a Flag-tagged acquired randomly from fields with untangled fibers us- full-length WRN plasmid carrying Ala substitutions at all ing Eclipse 80i Nikon Fluorescence Microscope, equipped the six S/TQ sites (WSWRN6A) were generated as previ- with a VideoConfocal (ViCo) system. The length of labeled ously described (10,12). tracks were measured using the Image-Pro-Plus 6.0 soft- All the cell lines were maintained in Dulbecco’s modi- ware, and values were converted into kilobase using the con- fied Eagle’s medium (DMEM; Life Technologies) supple- version factor 1 ␮m = 2.59 kb as reported (19). A minimum mented with 10% Fetal bovine serum (FBS) (Boehringer ◦ of 100 individual fibers were analyzed for each experiment Mannheim) and incubated at 37 C in a humidified 5% CO 2 and the mean of at least three independent experiments pre- atmosphere. sented. Plasmids and transfection Statistical analysis Plasmids expressing the Flag-tagged WRNwt or the Flag- All the data are presented as means of at least three indepen- tagged WRN6A were generated as earlier reported (12). dent experiments. Statistical comparisons of WS or WRN- The phospho-mimic (Flag-CHK1317/345D) mutant form of mutant cells to their relevant control were analyzed by Stu- CHK1, a kind gift from Professor K.K. Khanna (Queens- dent’s t-test. P < 0.0001 was considered significant. land Institute of Medical Research, Australia), was con- structed as previously described (17). To express the plas- RESULTS mids, cells were transfected using the NeonTM Transfection WRN deficiency results in defective ATR-dependent check- System Kit (Invitrogen), according to the manufacturer’s in- point activation under mild replication stress structions. To assess the role for WRN in ATR pathway activation in response to mild replication stress, we examined the Immunoprecipitation, cell fractionation and western blot phosphorylation status of the main target of ATR, CHK1. analysis To compare isogenic cell lines, HEK293T cells stably ex- Immunoprecipitation and chromatin fractionation exper- pressing scrambled (WRN-wt) or WRN-targeting shRNA iments were performed as previously described (12,18). (WRN-kd) were generated. WRN-kd cells showed about Quantification was performed on scanned images of blots 80% depletion of WRN protein under the experimental con- using ImageJ software, and values shown on the graphs rep- ditions used in this study (Figure 1A). Treatment with low resent a percentage compared with the matched untreated dose of Aph induced a time-dependent phosphorylation of control normalized against the protein content evaluated CHK1 in WRN-wt cells, already noticeable after 1 h and through Lamin B1 immunoblotting. peaking at 24 h (Figure 1A), suggesting that also a modest Cell lysates were prepared as previously reported (18). replication perturbation can trigger a quick checkpoint re- Antibodies used for western blot (WB) were commer- sponse. In contrast, CHK1 phosphorylation was absent, or cially obtained for Flag-Tag (Sigma-Aldrich), phospho- very weak, in WRN-kd cells, and it was detectable only at S/TQ, phospho-CHK1-Ser345 (Cell Signaling Technolo- the late time-points even if not at the wild-type levels (Fig- gies), WRN, CHK1, Cyclin A, RAD9 (Santa Cruz Biotech- ure 1A). However, unlike the nanomolar dose of Aph, treat- nology, Inc.), Lamin B1 (Abcam), GAPDH (EMD Mil- ment with 1 mM HU, which leads to a robust genome-wide lipore), RPA32 (Calbiochem),
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