DNA Damage Checkpoint Activation Research Articles

Tryptophanyl-tRNA synthetase-1 (WARS-1) depletion and high tryptophan concentration lead to genomic instability in Caenorhabditis elegans

The fidelity of translation is ensured by a family of proteins named aminoacyl-tRNA synthetases (ARSs), making them crucial for development and survival. More recently, mutations in the tryptophanyl-tRNA synthetase 1 (WARS1) have been linked to various human diseases, from intellectual disability to various types of cancer. To understand the function of WARS1, we investigated the effect of WARS-1 depletion during the mitotic and meiotic cell cycle in the developing germline of Caenorhabditis elegans (C. elegans) and demonstrated the role of WARS-1 in genome integrity. wars-1 knockdown results in cell cycle arrest of the mitotically active germ cells. Such mitotic arrest is also associated with canonical DNA damage-induced checkpoint signaling in mitotic and meiotic germ cells. Significantly, such DNA checkpoint activation is associated with the morphological anomalies in chromatin structures that are the hallmarks of genome instability, such as the formation of chromatin bridges, micronuclei, and chromatin buds. We demonstrated that knocking down wars-1 results in an elevation of the intracellular concentration of tryptophan and its catabolites, a surprising finding emphasizing the impact of cellular amino acid availability and organismal/individual dietary uptake on genome integrity. Our result demonstrates that exposing C. elegans to a high tryptophan dosage leads to DNA damage checkpoint activation and a significant increase in the tryptophan metabolites. Targeting tryptophan catabolism, the least utilized amino acid in nature, can be important in developing new cancer therapeutic approaches. All in all, we have strong evidence that knocking down wars-1 results in defects in genomic integrity.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms. This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast.

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

MiR-669b-5p inhibits the Alzheimer's disease development via regulation of CHEK2 in Neuro2a APPSwe/Δ9 cells.

DNA damage of neurons is accumulated in Alzheimer's disease (AD). DNA damage-activated Checkpoint kinase 2 (CHEK2) is evaluated in Aβ-treated Neuro2a APPSwe/Δ9 cells, and the miR-669b-5p was specifically down-regulated. However, the underlying molecular mechanism between CHEK2 and miR-669b-5p in Neuro2a APPSwe/Δ9 cells remains unclear. This research discovers that in A-treated Neuro2a APPSwe/Δ9 cells, CHEK2 expression and miR-669b-5p expression were inversely correlated. In addition, miR-669b-5p mimics increased cell survival and proliferation in Neuro2a APPSwe/Δ9 cells while decreasing the production of inflammatory cytokines and cell death. Furthermore, it is observed that CHEK2 was a miR-669b-5p downstream target gene and that CHEK2 restored the miR-669b-5p's functions. According to this research, miR-669b-5p is a potential therapy for Alzheimer's patients since it slows the advancement of the disease.

Polyethylene nanoplastics cause reproductive toxicity associated with activation of both estrogenic hormone receptor NHR-14 and DNA damage checkpoints in C. elegans

As the most commercial polymer, the polyethylene nanoparticle (PE-NP) has been discharged into the environment and poses potential risks to organisms. However, the possible reproductive toxicity of PE-NP and underlying mechanisms remain largely unknown. In this study, Caenorhabditis elegans was employed as the animal model to effects of PE-NP (100 nm) and their leachates on reproduction and underlying mechanisms. Nematodes were exposed to PE-NP at 0.1–100 μg/L from L1-larvae to adult day 1 (approximately 4.5 days). Both brood size and number of fertilized eggs in uterus were decreased by 10 and 100 μg/L PE-NP, but could not be affected by their leachates. In addition, number of mitotic cells, length, and area of gonad were reduced by 10 and 100 μg/L PE-NP, but were not altered by their leachates. Accompanied with alteration in expressions of genes (egl-1, ced-9, ced-4, and ced-3) governing cell apoptosis, germline apoptosis was enhanced by PE-NP. Meanwhile, DNA damage was involved in the enhancement germline apoptosis after PE-NP exposure. PE-NP further increased expression of nhr-14 encoding estrogenic hormone receptor, and RNAi of nhr-14 suppressed PE-NP reproductive toxicity. Moreover, RNAi of nhr-14 decreased expression of egl-1, ced-4, ced-3, and mrt-2 in PE-NP exposed nematodes. Therefore, exposure to PE-NPs rather than in their leachates potentially caused reproductive toxicity by activating both estrogenic hormone receptor NHR-14 and DNA damage checkpoints (CLK-2, HUS-1, and MRT-2) in nematodes. These findings provide important insights into the exposure risk of PE-NPs on reproduction of environmental organisms.

Epigenetic instability caused by absence of CIZ1 drives transformation during quiescence cycles

BackgroundCip1-interacting zinc finger protein 1 (CIZ1) forms RNA-dependent protein assemblies that stabilise epigenetic state, notable at the inactive X chromosome in females. CIZ1 has been linked with a range of human cancers and in mice genetic deletion of CIZ1 manifests as hyperproliferative lymphoid lineages in females. This suggests that its role in maintenance of epigenetic stability is linked with disease.ResultsHere, we show that male and female CIZ1-null primary murine fibroblasts have reduced H4K20me1 and that this compromises nuclear condensation on entry to quiescence. Global transcriptional repression remains intact in condensation-deficient CIZ1-null cells; however, a subset of genes linked with chromatin condensation and homology-directed DNA repair are perturbed. Failure to condense is phenotypically mimicked by manipulation of the H4K20me1 methyltransferase, SET8, in WT cells and partially reverted in CIZ1-null cells upon re-expression of CIZ1. Crucially, during exit from quiescence, nuclear decondensation remains active, so that repeated entry and exit cycles give rise to expanded nuclei susceptible to mechanical stress, DNA damage checkpoint activation, and downstream emergence of transformed proliferative colonies.ConclusionsOur results demonstrate a role for CIZ1 in chromatin condensation on entry to quiescence and explore the consequences of this defect in CIZ1-null cells. Together, the data show that CIZ1’s protection of the epigenome guards against genome instability during quiescence cycles. This identifies loss of CIZ1 as a potentially devastating vulnerability in cells that undergo cycles of quiescence entry and exit.

CDT1 is the major functional regulatory subunit of the pre-replication complex in zygotes.

Pre-replication complex (pre-RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre-RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re-replication and thus DNA damage check-point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre-RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its' degradation is essential for zygotes to prevent from DNA re-replication in G2 stage.

Tolerance to replication stress requires Dun1p kinase and activation of the electron transport chain

One of the key outcomes of activation of DNA replication checkpoint (DRC) or DNA damage checkpoint (DDC) is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), which is a prerequisite for normal progression through the S phase and for effective DNA repair.We have recently shown that DDC increases aerobic metabolism and activates the electron transport chain (ETC) to elevate ATP production and dNTP synthesis by repressing transcription of histone genes, leading to globally altered chromatin architecture and increased transcription of genes encoding enzymes of tricarboxylic acid (TCA) cycle and the ETC. The aim of this study was to determine whether DRC activates ETC. We show here that DRC activates ETC by a checkpoint kinase Dun1p-dependent mechanism. DRC induces transcription of RNR1-4 genes and elevates mtDNA copy number. Inactivation of RRM3 or SGS1, two DNA helicases important for DNA replication, activates DRC but does not render cells dependent on ETC. However, fitness of rrm3Δ and sgs1Δ cells requires Dun1p. The slow growth of rrm3Δdun1Δ and sgs1Δdun1Δ cells can be suppressed by introducing sml1Δ mutation, indicating that the slow growth is due to low levels of dNTPs. Interestingly, inactivation of ETC in dun1Δ cells results in a synthetic growth defect that can be suppressed by sml1Δ mutation, suggesting that ETC is important for dNTP synthesis in the absence of Dun1p function. Together, our results reveal an unexpected connection between ETC, replication stress, and Dun1p kinase.

Mechanisms on chemotherapy resistance of colorectal cancer stem cells and research progress of reverse transformation: A mini-review.

Tumor recurrence and chemotherapy resistance are mainly responsible for poor prognosis in colorectal cancer (CRC) patients. Cancer stem cell (CSC) has been identified in many solid tumors, including CRC. Additionally, CSC cannot be completely killed during chemotherapy and develops resistance to chemotherapeutic drugs, which is the main reason for tumor recurrence. This study reviews the main mechanisms of CSC chemotherapy resistance in CRC, including activation of DNA damage checkpoints, epithelial-mesenchymal transition (EMT), inhibition of the overexpression of antiapoptotic regulatory factors, overexpression of ATP-binding cassette (ABC) transporters, maintenance of reactive oxygen species (ROS) levels, and the dormant state of CSC. Advances in research to reverse chemotherapy resistance are also discussed. Our study can provide the promising potential for eliminating CSC and preventing tumor progression for CRC treatment.

Exploring the molecular and functional cellular response to hydrazine via transcriptomics and DNA repair mutant cell lines.

Hydrazine is a rodent carcinogen and is classified as a probable human carcinogen by IARC. Though hydrazine is positive in both in vitro and in vivo DNA strand break (comet) assays, hydrazine was reported to be negative in an in vitro mutation Muta Mouse lung epithelial cell (FE1) test, as well as in a regulatory-compliant, in vivo Big Blue mouse mutation test. In this article, mechanistic studies explored the cellular response to hydrazine. When tested in a regulatory-compliant mouse lymphoma assay, hydrazine yielded unusual, weakly positive results. This prompted an investigation into the transcriptional response to hydrazine in FE1 cells via RNA sequencing. Amongst the changes identified was a dose-dependent increase in G2/M DNA damage checkpoint activation associated genes. Flow cytometric experiments in FE1 cells revealed that hydrazine exposure led to S-phase cell cycle arrest. Clonogenic assays in a variety of cell lines harboring key DNA repair protein deficiencies indicated that hydrazine could sensitize cells lacking homology dependent repair proteins (Brca2 and Fancg). Lastly, hprt assays with hydrazine were conducted to determine whether a lack of DNA repair could lead to mutagenicity. However, no robust, dose-dependent induction of mutations was noted. The transcriptional and cell cycle response to hydrazine, coupled with functional investigations of DNA repair-deficient cell lines support the inconsistencies noted in the genetic toxicology regulatory battery. In summary, while hydrazine may be genotoxic, transcriptional and functional processes involved in cell cycle regulation and DNA repair appear to play a nuanced role in mediating the mutagenic potential.

R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline.

Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.

When more is less: heritable gain-of-function chk1 mutations impair human fertility.

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.

The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells

Triple-negative breast cancer (TNBC) lacks significant expression of the estrogen receptor, the progesterone receptor, and of human epidermal growth factor receptor. It is the most aggressive and malignant of all breast cancers, and for which, there are currently no effective targeted therapies. We have shown previously that the RecQ helicase family member RECQL5 is essential for the proliferation and survival of TNBC cells; however, the mechanism of its involvement in cell viability has not been shown. Here, we report that the expression of RecQ family helicases, including RECQL5, is regulated by the deubiquitinase USP28. We found using genetic depletion or a small molecule inhibitor that like RECQL5, USP28 is also essential for TNBC cells to proliferate in vitro and in vivo. Compromising the function of USP28 by shRNA knockdown or the inhibitor caused TNBC cells to arrest in S/G2 phases, concurrent with DNA-damage checkpoint activation. We further showed that the small molecule inhibitor of USP28 displayed anti-tumor activity against xenografts derived from TNBC cells. Our results suggest that USP28 could be a potential therapeutic target for triple negative breast cancer.

Rad50 promotes ovarian cancer progression through NF-κB activation.

Rad50 is a component of MRN (Mre11‐Rad50‐Nbs1), which participates in DNA double‐strand break repair and DNA‐damage checkpoint activation. Here, we sought to investigate the clinical and functional significance of Rad50 in high‐grade serous ovarian cancer (HGSOC). We found that Rad50 was frequently upregulated in HGSOCs and enhanced Rad50 expression inversely correlated with patient survival. In addition, ectopic expression of Rad50 promoted proliferation/invasion and induced EMT of ovarian cancer cells, whereas knockdown of Rad50 led to decreased aggressive behaviors. Mechanistic investigations revealed that Rad50 induced aggressiveness in HGSOC via activation of NF‐κB signaling pathway. Moreover, we identified CARD9 as an interacting protein of Rad50 in ovarian cancer cells and the activation of NF‐κB pathway by Rad50 is CARD9 dependent. Our findings provide evidence that Rad50 exhibits oncogenic property via NF‐κB activation in HGSOC.

DNA damage checkpoint activation affects peptidoglycan synthesis and late divisome components in Bacillus subtilis.

During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

Set1-dependent H3K4 methylation becomes critical for limiting DNA damage in response to changes in S-phase dynamics in Saccharomyces cerevisiae

DNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S-phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. The opposite effects of the lack of RNase H activity and the reduction of histone levels on orc5-1 set1Δ viability are in agreement with their expected effects on replication fork progression. We propose that the role of H3K4 methylation during DNA replication becomes critical when the replication forks acceleration due to decreased origin firing in the orc5-1 background increases the risk for transcription replication conflicts. Furthermore, we show that an increase of reactive oxygen species levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions, Telomere Dysfunction and Inhibition of DNA Repair.

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.