Unreplicated DNA Research Articles

Monoubiquitinated H2B, a Main Chromatin Target of Formaldehyde, Is Important for S-Phase Checkpoint Signaling and Genome Stability.

Formaldehyde (FA) is a human carcinogen with ubiquitous environmental exposures and significant endogenous formation. Genotoxic activity of FA stems from its reactivity with DNA-NH2 groups. Histone lysines are another source of aldehyde-reactive amino groups in chromatin, however, chromatin/histone damage responses to FA and their biological significance are poorly understood. We examined histone posttranslational modifications in FA-treated human lung cells and found that the majority of the most prominent small lysine modifications associated with active or inactive chromatin were unchanged. FA moderately decreased H3K9 and H3K27 acetylation and H2A-K119 monoubiquitination but caused surprisingly severe losses of H2B-K120 monoubiquitination, especially in primary and stem-like cells. H2Aub1 decreases reflected its slower ubiquitination linked to a lower ubiquitin availability due to K48-polyubiquitination of FA-damaged proteins. Depletion of H2Bub1 resulted from its rapid deubiquitination in part by ATXN7L3-associated deubiquitinases and was independent on DNA damage signaling, indicating a direct chromatin damage response. Manipulations of H2Bub1 abundance showed that it was important for robust ATM and ATR signaling, efficient S-phase checkpoint, and suppression of mitotic transmission of unreplicated DNA and formation of micronuclei. Our findings identified H2B deubiquitination as a major FA-induced chromatin damage response that regulates S-phase checkpoint signaling and genome stability.

PCNA recruits cohesin loader Scc2 to ensure sister chromatid cohesion

Sister chromatid cohesion, established during replication by the ring-shaped multiprotein complex cohesin, is essential for faithful chromosome segregation. Replisome-associated proteins are required to generate cohesion by two independent pathways. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly replicated DNA. The latter process depends on the cohesin loader Scc2 (NIPBL in vertebrates) and the alternative PCNA loader CTF18-RFC. However, the mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits the yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as the scc2-pip mutant deficient in Scc2–PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells.

Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation.

Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered ‘heavy-light’ (HL) by incorporation of BrdUTP while unreplicated DNA remains ‘light-light’ (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.

CHK1 inhibition exacerbates replication stress induced by IGF blockade

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

Locus-specific transcription silencing at the FHIT gene suppresses replication stress-induced copy number variant formation and associated replication delay.

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.

Telomere Replication: Solving Multiple End Replication Problems.

Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

SummaryLesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase–anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

Replication stress limits developmental potential of human preimplantation embryos

Human preimplantation embryos show abnormal nucleation and DNA damage, compromising cell cycle progression and developmental potential. The molecular mechanisms and timing of these abnormalities are unknown. We identified endogenous DNA damage and repair mechanisms in human embryos and developed a murine model to study developmental consequences of replication stress in preimplantation embryos. Prospective laboratory study of human and mouse embryos Endogenous DNA damage and repair pathways were evaluated in humans using donated oocytes (n=25) and embryos (n=20). Immunofluorescent staining detected DNA damage (γH2AX, RPA), repair (RPAS33, RPA S4/S8, Rad51, 53BP1), and micronucleation. Consequences of genomic instability were studied in a murine model using aphidicolin, a DNA polymerase inhibitor that increases endogenous chromosome fragility1. Mouse zygotes (n=500) were briefly exposed to aphidicolin in the first cell cycle, then evaluated at 1-cell, 2-cell, 4-8-cell and blastocyst stage. Immunofluorescent staining detected DNA damage and repair. Cleavage progression, blastulation and embryo quality were assessed versus controls. Human and mouse karyotypes were determined with NGS. Human preimplantation embryos show endogenous DNA damage, demonstrated by γH2AX, RPA and abnormal nucleation. Cleavage embryos had significantly greater foci and micronucleation vs blastocysts (γH2AX cleavage mean 2.3 vs blastocyst 1.0, p <0.0001; RPA cleavage mean 1.7 vs 0.3, p<0.0001; abnormal nucleation cleavage mean 15.9% vs blastocyst 4.2%, p<0.0001). DNA damage foci coincided with RPAS33, indicating RPA phosphorylation by G2 checkpoint kinase ATR, Rad51, indicating repair by homologous recombination, and 53BP1, indicating unrepaired DNA is passed to daughter cells. Aphidicolin-induced replication delay resulted in DNA damage (γH2AX and RPA), and RPAS33, indicating an ATR-dependent G2 checkpoint. Additional DNA repair mechanisms included Rad51 and 53BP1, similar to human embryos. Though some unreplicated DNA is tolerated in mitosis and compatible with euploidy, aphidicolin-induced under replication in the first cell cycle precipitated instability in later cell cycles, leading to decreased blastulation (45% after 8h aphidicolin vs 91.8% control, p<0.0001), and poor quality embryos as evidenced by significantly fewer total cells and inner cell mass with significantly greater DNA damage and micronucleation with increasing duration of aphidicolin exposure compared to controls. DNA damage responses to incomplete replication in G2 (ATR and Rad51), and the G1 response to unreplicated DNA (53BP1) mirror endogenous repair activity in human preimplantation embryos. Developmental consequences of replication stress likely persist beyond the preimplantation stage and may contribute to failed implantation or miscarriage. The murine model of genomic instability enables further study of these processes and the development of targeted therapeutics.

Genome sizes and genomic guanine+cytosine (GC) contents of the Czech vascular flora with new estimates for 1700 species

The content of DNA in the somatic, unreplicated cell nuclei (genome size) and DNA base composition (GC content) are the basic genomic parameters that can be measured by flow cytometry. Genome size, or ploidy level, can affect many plant properties and are therefore included as important features in modern biological floras and plant trait databases. However, genomic data are still largely measured mainly for taxonomic and biosystematic purposes, and despite the popularity of flow cytometry in the Czech Republic, this information is still missing for many of the vascular plants in the Czech flora. The biological significance of the GC content is less clear compared to the genome size, which, along with the greater difficulty in measuring it, results in the absence of such information for the vastmajority of species. Here, we measure these two genomic parameters for 1908 samples of 1700 species in the Czech vascular flora. Here for the first time are reported the genome sizes of more than 1000 species and GC contents of more than 1500 species, which more than doubles the amount of information on the GC content of vascular plants. Together with the published data obtained in our laboratory using the same methods and flow cytometers, this information is now available for 1910 species that occur in the Czech Republic (~83% of this country’s permanent flora, excluding apomicts). They are summarized in a table, accompanied by information about assumed chromosome number, ploidy level and an estimate of the monoploid genome size.We further provide a descriptive analysis of this dataset, list extreme values and comment on some cytogeographically interesting findings. This dataset is the largest and most comprehensive set of genomic data covering almost the entire flora of a country. It will serve as the basis of the karyological traits section of the Czech plant trait database Pladias (www.pladias.cz) and for testing of hypotheses about the evolution and biological relevance of these genomic parameters.

Genome-wide stability of the DNA replication program in single mammalian cells.

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.

Analysis of Cohesin Association to Newly Replicated DNA Through Nascent Strand Binding Assay (NSBA).

Replication forks engage chromatin-bound cohesin complexes during chromosome replication. Interfacing between cohesin and replication forks influences both cohesion establishment and fork functionality. However, the mechanisms mediating this process are scarcely understood. Here we describe the nascent strand binding assay (NSBA) methodology, developed in budding yeast to discriminate the association of cohesin to either parental unreplicated or nascent DNA in the environment of replication forks. NSBA quantitatively estimates the association of a protein of interest to newly replicated DNA. For this, nascent strands are in vivo labeled with the thymine analogue bromodeoxyuridine and chromatin is immunoprecipitated to isolate a fraction enriched in DNA associated to the target protein. The abundance of nascent DNA is then assessed through BrdU immunoprecipitation followed by quantitative PCR, allowing for the parallel analysis of diverse genomic regions. While originally employed to characterize the association of cohesin to nascent sister chromatids, NSBA can be applied to study other factors dynamically associating to nascent DNA.

TIAR marks nuclear G2/M transition granules and restricts CDK1 activity under replication stress

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA. Here, we show that the RNA‐binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

The time-dependent rate [Formula: see text] of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here, we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the [Formula: see text] universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the [Formula: see text] maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.

Cohesin dynamic association to chromatin and interfacing with replication forks in genome integrity maintenance.

Proliferating cells need to accurately duplicate and pass their genetic material on to daughter cells. Problems during replication and partition challenge the structural and numerical integrity of chromosomes. Diverse mechanisms, as the DNA replication checkpoint, survey the correct progression of replication and couple it with other cell cycle events to preserve genome integrity. The structural maintenance of chromosomes (SMC) cohesin complex primarily contributes to chromosome duplication by mediating the tethering of newly replicated sister chromatids, thus assisting their equal segregation in mitosis. In addition, cohesin exerts important functions in genome organization, gene expression and DNA repair. These are determined by cohesin's ability to bring together different DNA segments and, hence, by the fashion and dynamics of its interaction with chromatin. It recently emerged that cohesin contributes to the protection of stalled replication forks through a mechanism requiring its timely mobilization from unreplicated DNA and relocation to nascent strands. This mechanism relies on DNA replication checkpoint-dependent cohesin ubiquitylation and promotes nascent sister chromatid entrapment, likely contributing to preserve stalled replisome-fork architectural integrity. Here we review how cohesin dynamic association to chromatin is controlled through post-translational modifications to dictate its functions during chromosome duplication. We also discuss recent insights on the mechanism that mediates interfacing of replisome components with chromatin-bound cohesin and its contribution to the establishment of sister chromatid cohesion and the protection of stalled replication forks.

Single-cell replication profiling to measure stochastic variation in mammalian replication timing

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments. Although replication origins are selected stochastically, variation in replication timing is poorly understood. Here we devise a strategy to measure variation in replication timing using DNA copy number in single mouse embryonic stem cells. We find that borders between replicated and unreplicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Fifty percent of replication events deviated from their average replication time by ± 15% of S phase. This degree of variation is similar between cells, between homologs within cells and between all domains genomewide, regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

Cohesin Can Remain Associated with Chromosomes during DNA Replication

SummaryTo ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.