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Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polα downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polα is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polα and RAD51 is stimulated by RAD52 inhibition, and Polα-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polα-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled.

PDF file, 1010K, Chemical structures of structurally related analogs of the previously identified parent compound NSC 19630 (S1); Effect of NSC 617145 on WRN catalytic functions and DNA unwinding by other helicases (S2); Effect of selected small molecules structurally related to NSC 19630 on HeLa cell proliferation (S3); NSC 617145 inhibits cell proliferation in a WRN-dependent manner (S4); Effect of NSC 617145 on WRN-depleted cells expressing ectopic WRN protein that is active or inactive as for ATPase/helicase activity (S5); Specificity of WRN helicase inhibition by NSC 617145 (S6); NSC 617145 induces DNA damage (S7); NSC 617145 induces apoptosis when WRN is present (S8); Elevated PCNA staining of NSC 617145-treated cells is WRNdependent (S9); Effect of NSC 617145 exposure on cell cycle (S10); NSC 617145 exposure does not sensitize HeLa, FA-A, or FA-D2 cells to hydroxyurea (S11); WRN depletion does not affect MMC sensitivity of FA-D2 mutant cells (S12); Western blot analysis of ATM activation in FA-D2 mutant cells (S13).

PDF file, 108K.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52–ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23–1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3–8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Maintenance of genome integrity is essential for cell viability and depends on complete and accurate DNA replication. However, G4/R-loops may provide significant obstacles for DNA replication, as they can cause collisions between replication fork and the transcription machinery. Hence, cells require mechanisms to counteract the presence of persistent G4/R-loops, most of which remain poorly understood. Here, we demonstrate an involvement of the Werner helicase-interacting protein 1 (WRNIP1) in preventing DNA damage induced by G4/R-loop-associated transcription-replication conflicts. We discovered that the ubiquitin-binding domain of WRNIP1 is required to efficiently avoid pathological persistence of G4/R-loops upon replication stress. Also, we observed that G4s reside within R-loops and that WRNIP1 colocalises with these structures. Furthermore, WRNIP1 plays a role in restarting replication from transcription-induced fork stalling. More importantly, we characterized the interplay between WRNIP1 and the DNA helicase FANCJ in counteracting R-loop-dependent G4 formation in response to replication stress. Collectively, our findings propose a mechanisms whereby WRNIP1, contributing to stabilise FANCJ to G4 sites, mitigates the G4/R-loop-mediated transcription-replication conflicts and protects against DNA damage accumulation.

MUS81 is a structure-specific endonuclease that processes DNA intermediates in mitosis and in S-phase following replication stress. MUS81 is crucial to cleave deprotected reversed forks in BRCA2-deficient cells. However, how MUS81 is regulated during replication stress in human cells remains unknown.Our study reveals that CHK2 binds the MUS81-EME2 complex and positively regulates formation of DSBs upon replication stress or in the absence of BRCA2. The association with MUS81 occurs through the FHA domain of CHK2 and is disabled by the Li-Fraumeni-associated mutation I157T. The CHK2-MUS81-EME2 complex forms downstream fork reversal and degradation, and phosphorylation of MUS81 at CHK2-targeted sites is crucial to introduce DSBs at deprotected replication forks ensuring the replication fork recovery in BRCA2-deficient cells.Collectively, our work sheds light into the regulation of the MUS81-EME2 complex and identifies a novel function of the ATM-CHK2 axis in the response to deprotected replication forks in the absence of BRCA2.

The MUS81 complex is crucial for preserving genome stability through resolution of branched DNA intermediates in mitosis and also for the processing of deprotected replication forks in BRCA2-deficient cells. Because of the existence of two different MUS81 complexes in mammalian cells that act in M or S-phase, whether and how the PARPi sensitivity of BRCA2-deficient cells is affected by loss of MUS81 function is unclear.Here, using a mutant of MUS81 that impairs its function in M-phase, we show that viability of BRCA2-deficient cells but not their PARPi sensitivity requires a fully-functional MUS81 complex in mitosis. In contrast, expression of a constitutively-active MUS81 is sufficient to confer PARPi resistance. From a mechanistic point of view, our data indicates that deregulated action of the mitotic active form of MUS81 in S-phase leads to the cleavage of stalled replication forks before their reversal, bypassing fork deprotection, and engaging a Polθ-dependent DSBs repair.Collectively, our findings describe a novel mechanism leading to PARPi resistance that involves unscheduled MUS81-dependent cleavage of intact, unreversed replication forks. Since this cleavage occurs mimicking the phosphorylated status of S87 of MUS81, our data suggest that hyperphosphorylation of this residue in S-phase might represent a novel biomarker to identify resistance to PARPi.

Prompt diagnosis of complex phenotypes is a challenging task in clinical genetics. Whole exome sequencing has proved to be effective in solving such conditions. Here, we report on an unpredictable presentation of Werner Syndrome (WRNS) in a 12-year-old girl carrying a homozygous truncating variant in RECQL2, the gene mutated in WRNS, and a de novo activating missense change in PTPN11, the major Noonan syndrome gene, encoding SHP2, a protein tyrosine phosphatase positively controlling RAS function and MAPK signaling, which have tightly been associated with senescence in primary cells. All the major WRNS clinical criteria were present with an extreme precocious onset and were associated with mild intellectual disability, severe growth retardation and facial dysmorphism. Compared to primary fibroblasts from adult subjects with WRNS, proband's fibroblasts showed a dramatically reduced proliferation rate and competence, and a more accelerated senescence, in line with the anticipated WRNS features occurring in the child. In vitro functional characterization of the SHP2 mutant documented its hyperactive behavior and a significantly enhanced activation of the MAPK pathway. Based on the functional interaction of WRN and MAPK signaling in processes relevant to replicative senescence, these findings disclose a unique phenotype likely resulting from negative genetic interaction.

Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA–RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.

Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

Understanding basic molecular mechanisms underlying the biology of cancer cells is of outmost importance for identification of novel therapeutic targets and biomarkers for patient stratification and better therapy selection. One of these mechanisms, the response to replication stress, fuels cancer genomic instability. It is also an Achille’s heel of cancer. Thus, identification of pathways used by the cancer cells to respond to replication-stress may assist in the identification of new biomarkers and discovery of new therapeutic targets. Alternative mechanisms that act at perturbed DNA replication forks and involve fork degradation by nucleases emerged as crucial for sensitivity of cancer cells to chemotherapeutics agents inducing replication stress. Despite its important role in homologous recombination and recombinational repair of DNA double strand breaks in lower eukaryotes, RAD52 protein has been considered dispensable in human cells and the full range of its cellular functions remained unclear. Very recently, however, human RAD52 emerged as an important player in multiple aspects of replication fork metabolism under physiological and pathological conditions. In this review, we describe recent advances on RAD52’s key functions at stalled or collapsed DNA replication forks, in particular, the unexpected role of RAD52 as a gatekeeper, which prevents unscheduled processing of DNA. Last, we will discuss how these functions can be exploited using specific inhibitors in targeted therapy or for an informed therapy selection.

Emerging data indicate that the reverse transcriptase (RT) protein encoded by LINE-1 transposable elements is a promising cancer target. Nonnucleoside RT inhibitors, e.g. efavirenz (EFV) and SPV122.2, reduce proliferation and promote differentiation of cancer cells, concomitant with a global reprogramming of the transcription profile. Both inhibitors have therapeutic anticancer efficacy in animal models. Here we have sought to clarify the mechanisms of RT inhibitors in cancer cells. We report that exposure of PC3 metastatic prostate carcinoma cells to both RT inhibitors results in decreased proliferation, and concomitantly induces genome damage. This is associated with rearrangements of the nuclear architecture, particularly at peripheral chromatin, disruption of the nuclear lamina, and budding of micronuclei. These changes are reversible upon discontinuation of the RT-inhibitory treatment, with reconsititution of the lamina and resumption of the cancer cell original features. The use of pharmacological autophagy inhibitors proves that autophagy is largely responsible for the antiproliferative effect of RT inhibitors. These alterations are not induced in non-cancer cell lines exposed to RT inhibitors. These data provide novel insight in the molecular pathways targeted by RT inhibitors in cancer cells.

Conflicts between replication and transcription are common source of genome instability and many factors participate in prevention or removal of harmful R-loops. Here, we demonstrate that a WRNIP1-mediated response plays an important role in counteracting accumulation of aberrant R-loops. Using human cellular models with compromised ATR-dependent checkpoint activation, we show that WRNIP1 is stabilised in chromatin and is needed for maintaining genome integrity by mediating the ATM-dependent phosphorylation of CHK1. Furthermore, we show that loss of WRN or ATR signalling leads to accumulation of R-loop-dependent parental ssDNA, which is covered by RAD51. We demonstrate that WRNIP1 chromatin retention is also required to stabilise the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall our findings reveal a novel function of WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication-transcription conflicts are resolved.

Human papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.

HPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication., Human papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.

The Schimke immuno-osseous dysplasia is an autosomal recessive genetic osteochondrodysplasia characterized by dysmorphism, spondyloepiphyseal dysplasia, nephrotic syndrome and frequently T cell immunodeficiency. Several hypotheses have been proposed to explain pathophysiology of the disease, however, the mechanism by which SMARCAL1 mutations cause the syndrome is elusive. Indeed, animal models of the disease are absent or useless to provide insight into the disease mechanism, since they do not recapitulate the phenotype. We generated a conditional knockdown model of SMARCAL1 in iPSCs to mimic conditions of cells with severe form the disease. Here, we characterize this model for the presence of phenotype linked to the replication caretaker role of SMARCAL1 using multiple cellular endpoints. Our data show that conditional knockdown of SMARCAL1 in human iPSCs induces replication-dependent and chronic accumulation of DNA damage triggering the DNA damage response. Furthermore, they indicate that accumulation of DNA damage and activation of the DNA damage response correlates with increased levels of R-loops and replication-transcription interference. Finally, we provide data showing that, in SMARCAL1-deficient iPSCs, DNA damage response can be maintained active also after differentiation, possibly contributing to the observed altered expression of a subset of germ layer-specific master genes. In conclusion, our conditional SMARCAL1 iPSCs may represent a powerful model where studying pathogenetic mechanisms of severe Schimke immuno-osseous dysplasia, thus overcoming the reported inability of different model systems to recapitulate the disease.

Schimke immuno-osseous dysplasia is an autosomal recessive genetic osteochondrodysplasia characterized by dysmorphism, spondyloepiphyseal dysplasia, nephrotic syndrome and frequently T cell immunodeficiency. Several hypotheses have been proposed to explain the pathophysiology of the disease; however, the mechanism by which SMARCAL1 mutations cause the syndrome is elusive. Here, we generated a conditional SMARCAL1 knockdown model in induced pluripotent stem cells (iPSCs) to mimic conditions associated with the severe form the disease. Using multiple cellular endpoints, we characterized this model for the presence of phenotypes linked to the replication caretaker role of SMARCAL1. Our data show that conditional knockdown of SMARCAL1 in human iPSCs induces replication-dependent and chronic accumulation of DNA damage triggering the DNA damage response. Furthermore, they indicate that accumulation of DNA damage and activation of the DNA damage response correlates with increased levels of R-loops and replication-transcription interference. Finally, we provide evidence that SMARCAL1-deficient iPSCs maintain active DNA damage response beyond differentiation, possibly contributing to the observed altered expression of a subset of germ layer-specific master genes. Confirming the relevance of SMARCAL1 loss for the observed phenotypes, they are prevented or rescued after re-expression of wild-type SMARCAL1 in our iPSC model. In conclusion, our conditional SMARCAL1 knockdown model in iPSCs may represent a powerful model when studying pathogenetic mechanisms of severe Schimke immuno-osseous dysplasia., Summary: This study reports the generation of an iPSC model to investigate phenotypic changes associated with loss of SMARCAL1, as found in severe forms of the genetic disease Schimke immuno-osseous dysplasia.

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.

Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication&ndash, transcription conflicts are handled.

Human papillomaviruses (HPV) are double stranded DNA viruses causative in a host of human diseases including several cancers. Following infection two viral proteins, E1 and E2, activate viral replication in association with cellular factors, and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous replication (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2 replicating DNA and regulates the level of replication. Here we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2 replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2 replicating DNA. We present a model in which E1-E2 replication turns on the DDR stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2 replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.ImportanceHPV16 is the major viral human carcinogen, responsible for between 3 and 4% of all cancers worldwide. Following infection this virus activates the DNA damage response (DDR) to promote its life cycle, and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall the results suggest a mechanism where SIRT1 deacetylation of WRN promotes its interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication.

Replication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination, a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End-resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for the end-resection and is instrumental to select the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases define a complex regulatory layer that is essential for correct long-range end-resection connecting it to repair by homologous recombination. We establish that long-range end-resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed to conclude long-range end-resection and support RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as molecular switch to allow a timely execution of end-resection and repair at replication-dependent DNA double-strand breaks.

Stabilisation of the stalled replication fork is crucial to prevent excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is a human RecQ helicase that participates in the processing and recovery of perturbed replication forks. WRN is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined, and little is known about an involvement in the response to perturbed replication. Recently, the WRN exonuclease has been linked to protection of stalled forks from MRE11-dependent degradation in response to clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells, thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed replication forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon nanomolar doses of camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such alternative mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Serine 87, which is essential to avoid accumulation of mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.AUTHOR SUMMARYCorrect progression of the molecular machine copying the chromosomes is threatened by multiple causes that induce its delay or arrest. Once the replication machinery is arrested, the cell needs to stabilise it to prevent DNA damage. Many proteins contribute to this task and the Werner’s syndrome protein, WRN, is one of them.Defining what happens to replication machineries when they are blocked is highly relevant. Indeed, destabilised replication machineries may form upon treatment with anticancer drugs and influence the efficacy of some of them in specific genetic backgrounds. We used cells that lack one of the two enzymatic functions of WRN, the exonuclease activity, to investigate the fate of destabilised replication machineries. Our data show that they are handled by a repair pathway normally involved in fixing DNA breaks but, in this case, recruited to deal with regions of the genome that are left unreplicated after their destabilisation. This alternative mechanism involves a protein, RAD51, which tries to copy DNA from the sister chromosome. In so doing, however, RAD51 produces a lot of DNA interlinking that requires upregulation of a complex, called MUS81/EME1, which resolves this interlinking prior cell division and prevents accumulation of mitotic defects and cell death.

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS., Author summary Chromosomes are DNA molecules that contain the genetic information. During replication, the two sister DNA molecules covered by proteins (sister chromatids) are held together by many copies of a ring-like protein complex named cohesin, in a process called sister-chromatid cohesion. Before a cell divides, the cohesin rings are removed from the two sister chromatids to allow their migration towards the opposite poles of the dividing mother cell. At the end of this process, the two daughter cells have inherited a complete set of chromosomes. Before the next cell division, chromosomes are duplicated with high speed and fidelity. This important task is performed by the DNA replication machinery, a sophisticated apparatus made of several enzymes and proteins. In the present study, we have demonstrated that DDX11 and Timeless, two subunits of the DNA replication machinery, recruit the cohesin rings to promote their stable binding to the newly duplicated chromosomes, that is the establishment of sister-chromatid cohesion. In human cells that were genetically engineered to reduce the level of DDX11, we observed that sister-chromatid cohesion was loosened and association of cohesin to chromosomes was reduced. Our experimental results contribute to our understanding of the molecular mechanisms underlying the functional coupling between DNA replication and sister-chromatid cohesion in human cells.

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

Stabilisation of stalled replication forks prevents excessive fork reversal and their pathological degradation, which can undermine genome integrity. Here we investigate a physiological role of RAD52 at stalled replication forks by using human cell models depleted of RAD52, a specific small-molecule inhibitor of the RAD52-ssDNA interaction, in vitro and single-molecule analyses. We demonstrate that RAD52 prevents excessive degradation of reversed replication forks by MRE11. Mechanistically, RAD52 binds to the stalled replication fork, promotes its occlusion and counteracts loading of SMARCAL1 in vitro and in vivo. Loss of the RAD52 function results in a slightly-defective replication restart, persistence of under-replicated regions and chromosome instability. Moreover, the RAD52-inhibited cells rely on RAD51 for completion of replication and viability upon replication arrest. Collectively, our data suggest an unexpected gatekeeper mechanism by which RAD52 limits excessive remodelling of stalled replication forks, thus indirectly assisting RAD51 and BRCA2 in protecting forks from unscheduled degradation and preventing genome instability., Stabilisation of stalled replication forks prevents excessive fork reversal and genome instability. Here authors reveal a RAD52-dependent replication fork protection mechanism.

SLX4 is a versatile protein serving as docking for multiple structure-specific endonucleases during DNA repair, however, little is known about its function at demised replication forks. Using RNAi or FA-P cells complemented with SLX4 mutants that abrogate interaction with MUS81 or SLX1, we show that SLX4 cooperates with MUS81 to introduce DSBs after replication stress but also counteracts pathological targeting of demised forks by GEN1. Such unexpected function of SLX4 is unrelated to interaction with endonucleases, but concerns the physical presence of the protein. Strikingly, ectopic expression of the Holliday junction-binding protein RuvA inhibits DSBs in SLX4-deficient cells by preventing GEN1 chromatin-association, and rescues proliferation and genome integrity upon replication stress. Altogether, our results indicate that SLX4 is crucial to prevent accidental processing of Holliday junction-like intermediates at demised forks also suggesting that spontaneous genome instability in FA-P cells may derive, at least partially, from unscheduled action of GEN1 in S-phase.

Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.

The acquisition of genomic instability is a triggering factor in cancer development, and common fragile sites (CFS) are the preferential target of chromosomal instability under conditions of replicative stress in the human genome. Although the mechanisms leading to CFS expression and the cellular factors required to suppress CFS instability remain largely undefined, it is clear that DNA becomes more susceptible to breakage when replication is impaired. The models proposed so far to explain how CFS instability arises imply that replication fork progression along these regions is perturbed due to intrinsic features of fragile sites and events that directly affect DNA replication. The observation that proteins implicated in the safe recovery of stalled forks or in engaging recombination at collapsed forks increase CFS expression when downregulated or mutated suggests that the stabilization and recovery of perturbed replication forks are crucial to guarantee CFS integrity.

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2−/− cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia–telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2−/− cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia–deficient tumors by their sensitization to DNA cross-linking agents is discussed. Cancer Res; 73(17); 5497–507. ©2013 AACR.

Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch., End-resection of double strand DNA breaks is essential for pathway choice between non-homologous end-joining and homologous recombination. Here the authors show that phosphorylation of WRN helicase by CDK1 is essential for resection at replication-related breaks.

Failure to protect and/or restart stalled replication forks contributes to genomic instability. Radiation-sensitive 51 (RAD51) recombinase defends stalled forks from nucleolytic attack, which otherwise can threaten their integrity. Recently, we have uncovered a novel and key function of Werner helicase interacting protein 1 (WRNIP1) as a fork-protective factor working in conjunction with RAD51 in response to 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, eLife digest Cells are constantly in danger of losing or scrambling critical genetic information because of DNA damage. To cope with this stress, cells have numerous DNA repair systems. One of these systems – homology-directed DNA repair – involves the proteins BRCA1 and BRCA2, which are often missing or defective in breast and ovarian cancers. The BRCA-deficient cancer cells can still survive, but become “addicted” to other DNA repair proteins – among them a protein called RAD52. It might be possible to kill these cancer cells using drugs that stop RAD52 from working. Such treatments would have the benefit of not harming normal healthy cells, as these cells contain working BRCA proteins and can survive without RAD52. It is not currently known exactly how RAD52 allows the BRCA-deficient cells to survive, but this probably depends on RAD52’s ability to bind to single strands of DNA. Small molecules that block the interaction between the RAD52 protein and DNA might therefore help to kill cancer cells. Hengel et al. developed a high throughput biophysical method to search through a large collection of small molecules to find those that prevent RAD52 from binding to DNA. The best potential drug leads were then tested in laboratory-grown human cells and using biophysical and biochemical techniques. Computational approaches were also used to model how these molecules block the interaction between RAD52 and DNA at the atomistic level. Hengel et al. then used the information about how the small molecules bind to RAD52 to perform further computational screening. This identified a natural compound that competes with single-stranded DNA to bind to RAD52. The activity of this molecule was then validated using biophysical methods. The methods used by Hengel et al. provide the foundation for further searches for new anticancer drugs. Future studies that employ the small molecule drugs identified so far will also help to determine exactly how RAD52 works in human cells and how it helps cancer cells to survive. DOI: http://dx.doi.org/10.7554/eLife.14740.002

Mismatch repair (MMR) is the major repair pathway for removal of errors occurring during replication, which is often inactive in human tumours. Because MMR can control the cellular response to the cytotoxic effects of some DNA damaging drugs, here we will discuss how the MMR status can influence the biological response to chemotherapy. In addition we illustrate how some features of MMR-defective tumours might be exploited for personalized therapy treatments.

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

Common fragile sites (CFS) are difficult-to-replicate genomic regions that show a high propensity to breakage following certain forms of DNA replication stress. Long considered a fascinating component of human chromosome structure, their relevance for biology is proven by the fact that they are frequently rearranged in cancer cells. Furthermore, CFS were found to be the preferential targets for genome instability in the early stages of human tumorigenesis. In recent years, much progress has been made in understanding the structural features of CFS and the mechanisms that monitor and regulate their integrity. From these studies it has emerged that the reason for their fragility may depend on the abnormal high-frequency of fork stalling events occurring at CFS during DNA replication. Consistently, the ATR-dependent checkpoint together with several proteins involved in response to replication fork stalling have been implicated in maintaining CFS stability. Furthermore, more recent findings propose that the scarcity of replication initiation events within CFS may contribute to their expression upon replication perturbation. This review will focus on the molecular determinants responsible for genomic instability at CFS and the systems used by cells to address this eventuality.