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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.

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

The WRN protein is vital for managing perturbed replication forks. Replication Protein A strongly enhances WRN helicase activity in specific in vitro assays. However, the in vivo significance of RPA binding to WRN has largely remained unexplored. We identify several conserved phosphorylation sites in the acidic domain of WRN targeted by Casein Kinase 2. These phosphorylation sites are crucial for WRN-RPA interaction. Using an unphosphorylable WRN mutant, which lacks the ability to bind RPA, we determine that the WRN-RPA complex plays a critical role in fork recovery after replication stress countering the persistence of G4 structures after fork stalling. However, the interaction between WRN and RPA is not necessary for the processing of replication forks when they collapse. The absence of WRN-RPA binding hampers fork recovery, causing single-strand DNA gaps, enlarged by MRE11, and triggering MUS81-dependent double-strand breaks, which require repair by RAD51 to prevent excessive DNA damage. Here the authors show that the DNA helicase WRN ‘s interaction with the single stranded DNA binding protein RPA is regulated through specific phosphorylation by Casein Kinase 2. This regulation is critical for resolving DNA replication stress caused by G-quadruplexes.

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. By dissecting the so far loosely defined roles of the Werner syndrome helicase at stalled replication forks, this study shows that two highly related kinases can differentially regulate specific WRN phosphorylation sites and distinct WRN functions at various stages after replication arrest.

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.

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. 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.

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.

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.

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.

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.