Abstract

Article23 July 2009free access PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination Helen E Bryant Helen E Bryant The Institute for Cancer Studies, University of Sheffield, Sheffield, UK Search for more papers by this author Eva Petermann Eva Petermann Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK Search for more papers by this author Niklas Schultz Niklas Schultz Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Ann-Sofie Jemth Ann-Sofie Jemth Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Olga Loseva Olga Loseva Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Natalia Issaeva Natalia Issaeva Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Fredrik Johansson Fredrik Johansson Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Serena Fernandez Serena Fernandez The Institute for Cancer Studies, University of Sheffield, Sheffield, UK Search for more papers by this author Peter McGlynn Peter McGlynn School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Thomas Helleday Corresponding Author Thomas Helleday Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Helen E Bryant Helen E Bryant The Institute for Cancer Studies, University of Sheffield, Sheffield, UK Search for more papers by this author Eva Petermann Eva Petermann Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK Search for more papers by this author Niklas Schultz Niklas Schultz Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Ann-Sofie Jemth Ann-Sofie Jemth Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Olga Loseva Olga Loseva Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Natalia Issaeva Natalia Issaeva Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Fredrik Johansson Fredrik Johansson Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Serena Fernandez Serena Fernandez The Institute for Cancer Studies, University of Sheffield, Sheffield, UK Search for more papers by this author Peter McGlynn Peter McGlynn School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Thomas Helleday Corresponding Author Thomas Helleday Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden Search for more papers by this author Author Information Helen E Bryant1,‡, Eva Petermann2,‡, Niklas Schultz3,‡, Ann-Sofie Jemth3, Olga Loseva3, Natalia Issaeva3, Fredrik Johansson3, Serena Fernandez1, Peter McGlynn4 and Thomas Helleday 2,3 1The Institute for Cancer Studies, University of Sheffield, Sheffield, UK 2Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK 3Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden 4School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK ‡These authors contributed equally to this work *Corresponding author. Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK. Tel.: +44 1865 617 324; Fax: +44 1865 857 127; E-mail: [email protected] The EMBO Journal (2009)28:2601-2615https://doi.org/10.1038/emboj.2009.206 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks. Introduction Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein that is activated by DNA-strand breaks; activation of PARP1 leads to automodification and modification of other acceptor proteins with poly(ADP-ribose) (PAR) polymers (Satoh and Lindahl, 1992). PARP1 protects DNA breaks and chromatin structure and recruits DNA repair and checkpoint proteins to sites of damage (Allinson et al, 2003; Ahel et al, 2008). Inhibition of PARP1 is synthetic lethal with defects in homologous recombination (HR) and is currently being tested as a monotherapy for heritable breast and ovarian cancers deficient in the BRCA1 or BRCA2 genes (Bryant et al, 2005; Farmer et al, 2005; Helleday et al, 2008). PARP2, another nuclear member of the PARP family with largely unknown function, shares homology with PARP1 and is also activated by DNA breaks (Ame et al, 1999). Embryonic knockout of either PARP1 or PARP2 is well tolerated; however, double knockout is embryonic lethal (Menissier de Murcia et al, 2003) suggesting that PARP1 and PARP2 can compensate for some of each other's functions. PARP activity has been found to be enhanced in replicating cells (Lehmann et al, 1974), in the vicinity of replication forks (Jump et al, 1979) and in newly replicated chromatin (Anachkova et al, 1989). In addition, PARP1 has been shown to interact with several DNA replication proteins, many of which were poly(ADP-ribosyl)ated (Simbulan et al, 1993; Simbulan-Rosenthal et al, 1996; Dantzer et al, 1998). It has, therefore, been postulated that PARP1 might be involved in mammalian DNA replication. After treatment with hydroxyurea (HU), which stalls replication forks by depleting dNTP pools, PARP-1−/− cells have been shown to display delayed progress from S into G2/M phase (Yang et al, 2004), and although the underlying molecular mechanisms are not yet clear, a role for PARP1 in the response to replication fork stalling has been suggested. The PARP proteins are unique to higher eukaryotes and there is no evidence of PARP activity in Saccharomyces cerevisiae or prokaryotes. Regulation of DNA replication has been recognised as an important mechanism for preventing carcinogenesis, as impaired replication fork progression and increased replication-dependent DNA damage were observed in early stages of tumour development (Bartkova et al, 2006; Di Micco et al, 2006). In particular, the efficient reactivation of stalled replication forks is considered essential to maintain faithful replication and genomic stability. In Escherichia coli, stalled or collapsed replication forks are reactivated by recombination-dependent or -independent pathways, catalysed by the RuvABC or PriA and PriC proteins, respectively (Heller and Marians, 2006). These proteins are not conserved in eukaryotes, and eukaryotic mechanisms of replication fork reactivation are not well characterised. In yeast, HU-induced fork stalling is reversible and forks only collapse in certain backgrounds, for example rad53 mutants (Lopes et al, 2001); it is only in those backgrounds that HR is triggered for repair (Meister et al, 2005). This is in contrast to higher eukaryotes, in which HU triggers HR directly in wild-type mammalian cells (Arnaudeau et al, 2001; Saintigny et al, 2001; Lundin et al, 2002), suggesting that recombination-dependent replication restart mechanisms might be important in higher eukaryotes. Stalled forks are likely to need processing before HR medicated restart can take place, and this is supported by the fact that the end-processing Mre11 protein relocates to stalled replication forks after replication inhibition (Robison et al, 2004; Hanada et al, 2007). Here, we show that PARP1 binds to and is activated at stalled replication forks to mediate recruitment of Mre11 to initiate the end processing required for replication restart and HR. Results PARP is activated on replication stalling and required for survival Several lines of evidence suggest that PARP is associated with replication forks (Lehmann et al, 1974; Jump et al, 1979; Anachkova et al, 1989; Simbulan et al, 1993; Simbulan-Rosenthal et al, 1996; Dantzer et al, 1998; Yang et al, 2004). Here, we wanted to test whether PARP1 itself is involved in the response to stalled replication forks. We treated PARP1−/− mouse embryonic fibroblasts (MEFs) or PARP-inhibited cells with HU, which depletes dNTP pools and stalls replication forks (Bianchi et al, 1986). We found that both PARP-inhibited and PARP1−/− cells are sensitive to increasing doses of HU as compared with wild-type cells (Figure 1A; Supplementary Figure S1), showing that PARP is required for survival of replication fork stalling and confirming the HU sensitivity in PARP1−/− MEFs reported earlier (Yang et al, 2004). To further understand the role of PARP in promoting survival to HU, we tested whether PARP activity is also triggered by replication fork stalling, which would indicate an active role for PARP in the response to stalled forks. We found that the products of PARP activity, PAR polymers, are formed in cells treated with HU (Figure 1B–D). It is well established that PARP is activated by DNA single-strand breaks (SSBs), produced directly or as a base excision repair intermediate (Satoh and Lindahl, 1992), and that this attracts SSB repair proteins for repair (El-Khamisy et al, 2003). When investigating the kinetics of PARP activation, we found that HU treatment triggers a much slower PARP response than treatment with the alkylating agent methylmethane sulphonate (MMS) (Figure 1E) and, this is likely to be because of the low number of active forks that can be stalled by HU treatment in an asynchronous cell population. Figure 1.PARP is activated at stalled replication forks and required for survival of HU-induced replication stalling. (A) Surviving fraction of AA8 hamster cells treated for 10 days with increasing doses of HU in the presence or absence of PARP inhibitors NU1025 (100 nM), 1,5-dihydroxyisoquinoline (ISQ; 0.6 mM) or 4-amino-1,8-NAP (100 μM). (B) Immunofluorescence staining for PAR in AA8 hamster cells treated for 24 h with or without 0.5 mM HU. DNA was counterstained with TO-PRO-3 iodide. Bar 10 μm. (C) Quantification of immunofluorescence staining above. Percentage of AA8 cells containing sites of PARP activity induced by a 24-h treatment with 0.5 mM HU. Differences are statistically significant (Student's t-test, P20 HU-induced Mre11 foci was reduced when PARP was inhibited (Figure 6D). Mre11 is involved in resecting DNA ends (Williams et al, 2008) and is critical for repair of collapsed replication forks (Dolganov et al, 1996; Costanzo et al, 2001); this resection is thought to be essential because it allows HR-induced restart of forks. Our data, therefore, suggest that PARP exerts an effect to attract or retain Mre11 at sites of stalling, thus promoting resection, which could in turn allow for HR-mediated restart. The portion of Mre11 foci not overlapping with PAR polymers may reflect sites of non-homologous end joining, which can also repair HU-induced DSBs (Saintigny et al, 2001; Lundin et al, 2002). Using RPA foci as a marker of the amount of ssDNA produced, we tested whether resection of DNA is dependent on PARP. We found that fewer HU-induced RPA foci form in PARP1−/− as compared with PARP1+/+ MEFs and that PARP inhibitor reduced the formation of HU-induced RPA foci in wild-type cells (Figure 6E and F), suggesting that PARP1 activation is required for efficient formation of ssDNA regions at stalled replication forks; this is consistent with a role for PARP in recruitment of Mre11 for resection of DNA. PARP1 and Mre11 work in the same pathway for restart of stalled replication forks Although PARP1 facilitates Mre11 recruitment to stalled forks and promotes ssDNA formation, it is not clear whether this is related to the role of PARP in replication restart. To investigate the interplay between PARP1 and Mre11 during the reactivation of stalled replication forks, we depleted Mre11 using siRNA (Figure 7A) and found that Mre11-depleted cells showed a similar defect in replication restart as PARP1-depleted cells (Figure 7B and C; see Supplementary Figure S5 for a more detailed analysis). Moreover, we found that