The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with crosslinked DNA. FANCs interact with other proteins involved in both DNA repair and Sphase checkpoint such as BRCA1, ATM and the RAD50/ MRE11/NBS1 (RMN) complex. We deciphered the previously undefined pathway(s) leading to the ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found that ICLs activate a branched pathway downstream of the ATR kinase: one branch depending on CHK1 activity and the other on the FANCs-RMN complex. The transient slow-down of DNA synthesis was abolished in cells lacking ATR, whereas CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. This study demonstrates a novel relationship between the FA pathway(s) and the ATR kinase.

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.

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse. INTRODUCTIONWerner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging (Salk et al., 1985) and early onset of various neoplasms, including different types of carcinomas and sarcomas (Goto et al., 1981;Hrabko et al., 1982;Sato et al., 1988). This disorder arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. One of the hallmarks of WS patients is the genomic instability as evidenced by the spontaneous chromosome anomalies and large deletions in many genes (Salk et al., 1985;Fukuchi et al., 1989). It has been demonstrated that WRN exhibits DNA unwinding activity (Gray et al., 1997;Suzuki et al., 1997) and exonuclease activity residing in the N-terminal region (Huang et al., 1998). The cellular function of WRN is still unclear. It has been proposed that helicases are required for various DNA metabolic activities, including progression of replication fork, segregation of newly replicated chromosomes, disruption of the nucleosome structure, DNA supercoiling, transcription, recombination, and repair (Duguet, 1997). In addition, it has been suggested that RecQ family of DNA helicases has an important role in the maintenance of genomic integrity during DNA replication . Studies from yeast show that DNA replication does not proceed normally in absence of RecQ helicase function (Stewart et al., 1997) and in Xenopus laevis the ortholog of WRN is absolutely required for proper formation of replication foci (Yan et al., 1998). Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al., 1998;Brosh et al., 1999;Kamath-Loeb et al., 2000). Furthermore, in yeast, the RecQlike proteins seem involved in ...

Failure to stabilize and properly process stalled replication forks results in chromosome instability, which is a hallmark of cancer cells and several human genetic conditions that are characterized by cancer predisposition. Loss of WRN, a RecQ-like enzyme mutated in the cancer-prone disease Werner syndrome (WS), leads to rapid accumulation of double-strand breaks (DSBs) and proliferating cell nuclear antigen removal from chromatin upon DNA replication arrest. Knockdown of the MUS81 endonuclease in WRN-deficient cells completely prevents the accumulation of DSBs after fork stalling. Also, MUS81 knockdown in WS cells results in reduced chromatin recruitment of recombination enzymes, decreased yield of sister chromatid exchanges, and reduced survival after replication arrest. Thus, we provide novel evidence that WRN is required to avoid accumulation of DSBs and fork collapse after replication perturbation, and that prompt MUS81-dependent generation of DSBs is instrumental for recovery from hydroxyurea-mediated replication arrest under such pathological conditions.

WRN is a member of the RecQ family of DNA helicases implicated in the resolution of DNA structures leading to the stall of replication forks. Fragile sites have been proposed to be DNA regions particularly sensitive to replicative stress. Here, we establish that WRN is a key regulator of fragile site stability. We demonstrate that in response to mild doses of aphidicolin, WRN is efficiently relocalized in nuclear foci in replicating cells and that WRN deficiency is associated with accumulation of gaps and breaks at common fragile sites even under unperturbed conditions. By expressing WRN isoforms impaired in either helicase or exonuclease activity in defective cells, we identified WRN helicase activity as the function required for maintaining the stability of fragile sites. Finally, we find that WRN stabilizes fragile sites acting in a common pathway with the ataxia telangiectasia and Rad3 related replication checkpoint. These findings provide the first evidence of a crucial role for a helicase in protecting cells against chromosome breakage at normally occurring replication fork stalling sites.

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.

In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress.

The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing.DOI: http://dx.doi.org/10.7554/eLife.14740.001