Oncogene (2012) 31, 2809–2823 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The RAD9–RAD1–HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling P Pichierri, S Nicolai, L Cignolo1, M Bignami and A Franchitto Department of Environment and Primary Prevention, Istituto Superiore di Sanita`, Rome, Italy The WRN protein belongs to the RecQ family of DNA preference toward substrates that mimic structures helicases and is implicated in replication fork restart, but associated with stalled replication forks (Brosh et al., how its function is regulated remains unknown. We show 2002; Machwe et al., 2006) and WS cells exhibit that WRN interacts with the 9.1.1 complex, one of enhanced instability at common fragile sites, chromo- the central factors of the replication checkpoint. This somal regions especially prone to replication fork interaction is mediated by the binding of the RAD1 stalling (Pirzio et al., 2008). How WRN favors recovery subunit to the N-terminal region of WRN and is of stalled forks and prevents DNA breakage upon instrumental for WRN relocalization in nuclear foci and replication perturbation is not fully understood. It has its phosphorylation in response to replication arrest. We been suggested that WRN might facilitate replication also find that ATR-dependent WRN phosphorylation restart by either promoting recombination or processing depends on TopBP1, which is recruited by the 9.1.1 intermediates at stalled forks in a way that counteracts complex in response to replication arrest. Finally, we unscheduled recombination (Franchitto and Pichierri, provide evidence for a cooperation between WRN and 2004; Pichierri, 2007; Sidorova, 2008). This hypothesis is 9.1.1 complex in preventing accumulation of DNA supported by our recent findings, indicating that loss of breakage and maintaining genome integrity at naturally WRN results in excessive formation of double-stranded occurring replication fork stalling sites. Taken together, DNA breaks (DSBs) at stalled forks, which are our data unveil a novel functional interplay between WRN subsequently repaired through recombination (Pirzio helicase and the replication checkpoint, contributing to et al., 2008). shed light into the molecular mechanism underlying the Maintenance of genome stability during DNA synth- response to replication fork arrest. esis requires the function of the replication checkpoint, Oncogene (2012) 31, 2809–2823; doi:10.1038/onc.2011.468; which ensures proper handling of stalled forks and published online 17 October 2011 avoids DSB formation at replication intermediates. The replication checkpoint response is under the control of Keywords: RecQ helicases; replication checkpoint; the ATR kinase that is recruited to stalled forks through genome instability; Werner syndrome ATRIP-mediated binding to RPA-coated stretches of ssDNA (Cimprich and Cortez, 2008). Full activation of the checkpoint response requires the presence of the RAD9/RAD1/HUS1 (9.1.1) complex, which is loaded Introduction onto chromatin independently of ATR/ATRIP and stimulates the ATR activity through recruitment of the Werner syndrome protein (WRN) is a member of the TopBP1 mediator protein (Cimprich and Cortez, 2008). RecQ family of DNA helicases. Mutations in the WRN Indeed, TopBP1 associates directly with the 9.1.1 gene lead to a human disorder the Werner syndrome complex and, once recruited to stalled forks, facilitates (WS), characterized by a high incidence of cancer the ATR/ATRIP-mediated phosphorylation of several (Martin and Oshima, 2000; Muftuoglu et al., 2008b) other checkpoint proteins, in particular the downstream and wide genomic instability manifested as chromoso- kinase CHK1 (Zou et al., 2002; Furuya et al., 2004; mal abnormalities (Muftuoglu et al., 2008b). Mounting Delacroix et al., 2007). In addition, the 9.1.1 complex evidence strongly supports the idea that WRN has a acts as docking station for other proteins that are critical role in the maintenance of genome stability relocalized to replication forks or DNA damage sites, through faithful rescue of stalled replication forks. At such as DNA polymerase b, k and the DNA glycosylase the biochemical level, WRN shows a remarkable NEIL1 (Kai and Wang, 2003; Toueille et al., 2004; Guan et al., 2007). However, no functional interaction Correspondence: Dr P Pichierri, Section of Experimental and of the 9.1.1 complex with proteins that are directly Computational Carcinogenesis, Istituto Superiore di Sanita` , Viale correlated to replication fork processing and recovery Regina Elena 299, Rome 00161, Italy. has been identified so far. E-mail: [email protected] Even though WRN is phosphorylated in an ATR- 1Current address: Sanford-Burnham Medical Research Institute, San Diego, CA, USA dependent manner during the response to replication fork Received 28 March 2011; revised 3 September 2011; accepted 6 arrest, loss of ATR activity does not abolish WRN September 2011; published online 17 October 2011 recruitment to chromatin (Pichierri et al., 2003), suggest- WRN and the 9.1.1 complex interaction P Pichierri et al 2810 ing that other checkpoint factors may directly associate 2004; Toueille et al., 2004; Lan et al.,2005;Harriganet al., with WRN and affect its function in response to 2006), treatment of RAD9-depleted cells with agents replication fork stalling. Interestingly, disruption of the producing cell cycle-independent breaks (etoposide) or 9.1.1 complex in mice results in the accumulation of DSBs base damage to DNA (bleomycin) did not affect WRN at stalled forks and enhances chromosome fragility at relocalization in nuclear foci (Figure 1c). Similarly, genomic regions considered to be the equivalent of the transfection of HeLa cells using a single siRAD9 human common fragile sites (Zhu and Weiss, 2007). oligonucleotide, designed to target a region different from Here, we investigated the functional and physical those targeted by the pool of four independent siRAD9 interaction between WRN and the 9.1.1 complex and sequences (see also ‘Materials and methods’), efficiently disclosed the essential role of 9.1.1 in WRN recruitment downregulated RAD9 protein and prevented formation of to stalled forks and in the regulation of its ATR- WRN foci specifically after replication arrest induced by mediated phosphorylation. HU (Supplementary Figure 1). Consistently with a specific requirement of the 9.1.1 complex in the formation of WRN foci, introduction of an RNAi-resistant RAD9 Results protein in cells transfected with RAD9 siRNA restored the ability of WRN to localize in nuclear foci after replication The 9.1.1 complex is required for WRN relocalization and arrest (Supplementary Figure 2). phosphorylation following replication fork stalling Since RAD9 is necessary for WRN recruitment, we It is well recognized that in response to DNA damage or next examined whether WRN is required for RAD9 foci replication fork stalling, WRN leaves the nucleolus and formation following replication stress. We downregulated redistributes to nuclear foci (Constantinou et al., 2000; WRN expression in HeLa cells (Figure 1d) and analyzed Franchitto and Pichierri, 2004). Since several proteins formation of RAD9 foci by immunofluorescence. Down- that participate in DNA repair and checkpoint response regulation of WRN in untreated cells resulted in a small are loaded onto the chromatin by the 9.1.1 complex, we fraction of nuclei showing RAD9 localization in foci investigated whether WRN recruitment to stalled forks (Figure 1e). The percentage and brightness of nuclei with could be similarly regulated. To this aim, expression of RAD9 foci enhanced following HU or CPT exposure the RAD9 subunit was downregulated by RNAi in irrespective of the presence of WRN and appeared even HeLa cells, leading to the disruption of the entire increased in siWRN-treated cells (Figure 1e). complex (Figure 1a). RNAi-treated cells were chal- We have previously reported that in response to lenged with hydroxyurea (HU) or camptothecin (CPT) replication arrest, WRN is phosphorylated by an ATR- and examined for the ability of WRN to form foci. The dependent mechanism and assembles into nuclear foci majority of WRN was detected in nucleoli in untreated even in cells over-expressing the kinase dead form of cells, whereas, after treatments, it relocalized to nuclear ATR (Pichierri et al., 2003). Consistently, timecourse foci in cells transfected with either empty vector (mock) analysis showed that WRN phosphorylation occurs early or control small interfering RNA (siRNA) (siCtrl), but after replication arrest when WRN begins to form nuclear not in RAD9-depleted cells (Figure 1b). foci (Supplementary Figures 3A and B). We then Although both WRN and RAD9 (that is, the 9.1.1 investigated the dependence of the ATR-dependent complex altogether) are implicated in DSB and base WRN phosphorylation on the 9.1.1 complex. To test this excision repair (Roos-Mattjus et al., 2003; Cheng et al., hypothesis, HeLa cells, in which RAD9 had been depleted Figure 1 Abrogation of RAD9 function impairs WRN subnuclear relocalization and phosphorylation after HU treatment. (a) Depletion of RAD9 by RNAi. HeLa cells were transfected with siRNAs directed against GFP (siCtrl) or RAD9 (siRAD9) and cell lysates were prepared at the indicated time before immunoblotting with anti-RAD9 and anti-RAD1 antibodies. Anti-WRN antibody was used as loading control. (b) WRN relocalization in HeLa cells transfected with Ctrl