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PubMed2006

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Adenosine Triphosphatases↗
PubMed2026

Hypertranscription caused by p53 deficiency triggers nucleotide insufficiency that induces replication stress and genomic instability.

p53 plays a central role in the DNA damage response, inducing repair, cell-cycle arrest or apoptosis. Its loss is associated with replication stress and genomic instability. While several underlying mechanisms were suggested, the primary triggers of catastrophic genomic events like chromothripsis, a known driver of tumorigenesis linked with p53 loss, are still unclear. Using p53-depleted epithelial cells and fibroblasts, as well as patient-derived fibroblasts with germline p53 variants that spontaneously undergo chromothripsis, we found that p53 loss causes hypertranscription and increased nucleotide consumption. The resulting nucleotide shortage induces replication stress, causing telomere dysfunction, micronuclei formation, and chromothripsis. These effects were rescued by nucleoside supplementation or normalization of transcription levels, demonstrating a causal link between transcriptional activity, nucleotide availability, and genome stability. Emerging chromothriptic clones displayed restored DNA replication, telomere stabilization, and extrachromosomal DNA, suggesting key features that support clonal selection. We identify nucleotide pool homeostasis as a critical p53 function that suppresses replication stress, prevents chromothripsis, and protects against early tumorigenesis.

Genomic Instability↗
PubMed2006

Production of reactive oxygen species in response to replication stress and inappropriate mitosis in fission yeast.

Previous studies have indicated that replication stress can trigger apoptosis-like cell death, accompanied (where tested) by production of reactive oxygen species (ROS), in mammalian cells and budding yeast (Saccharomyces cerevisiae). In mammalian cells, inappropriate entry into mitosis also leads to cell death. Here, we report similar responses in fission yeast (Schizosaccharomyces pombe). We used ROS- and death-specific fluorescent stains to measure the effects of mutations in replication initiation and checkpoint genes in fission yeast on the frequencies of ROS production and cell death. We found that certain mutant alleles of each of the four tested replication initiation genes caused elevated ROS and cell death. Where tested, these effects were not enhanced by checkpoint-gene mutations. Instead, when cells competent for replication but defective in both the replication and damage checkpoints were treated with hydroxyurea, which slows replication fork movement, the frequencies of ROS production and cell death were greatly increased. This was a consequence of elevated CDK activity, which permitted inappropriate entry into mitosis. Thus, studies in fission yeast are likely to prove helpful in understanding the pathways that lead from replication stress and inappropriate mitosis to cell death in mammalian cells.

Cell Cycle Proteins↗
PubMed2026

CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress.

Eukaryotic cell division is regulated by CDK activity that must reach critical CDK threshold levels to progress through cell cycle stages. In low-mitogen, low-CDK environments, cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation promotes genomic instability after release from quiescence. Here, we show that CIZ1 contributes to mechanisms that temporally regulate cell cycle transitions in post-quiescent cells. CIZ1-/- (CIZ1 KO) fibroblasts re-entering the cell cycle from quiescence have reduced G1 phase and cell cycle length, mediated by increased intracellular CDK activity and early restriction point bypass via G1/S cyclin overexpression. In addition, CIZ1-/- cells are deficient in cyclin A chromatin binding and require increased CDK activity to initiate DNA replication, leading to DNA replication stress. Importantly, ectopic expression of CIZ1 or addition of recombinant CIZ1 reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. These data suggest that in post-quiescent cells, CIZ1 determines the threshold CDK activity required for the G1/S transition to prevent DNA replication stress.

DNA Replication↗
PubMed2026

ATR-dependent phosphorylation of the histone acetyltransferase HBO1 suppresses chromatin binding and promotes replication stress responses.

Mounting evidence has shown that histone acetyltransferase binding to ORC1 (HBO1) serves as an oncoprotein, warranting the use of the small molecule inhibitor WM-3835 for cancer therapy. However, HBO1 is ubiquitously expressed in both tumor and normal tissues, with potential to increase the risk of systemic toxicity. This unmet need highlights the importance of identifying suitable biomarkers to predict the sensitivity to HBO1 inhibitor. Here, we show that ATR, a key regulator of DNA replication stress, is a novel interacting partner of HBO1. In addition, we reveal a regulatory function of HBO1 in DNA replication stress responses, in an ATR-dependent manner. Mechanistically, ATR mediated HBO1 Ser50/53 phosphorylation interferes with the genomic binding of HBO1 and regulates gene expression. Notably, overexpression of HBO1 mutated at the ATR phosphorylation site (S50/53A) dampens the expression of DNA repair related genes and suppresses tumor colony formation, consistent with the observations of WM-3835 treatment. Inhibition of ATR significantly antagonized the sensitivity to WM-3835 treatment. Collectively, our findings uncovered a previously unidentified role of HBO1 in the regulation of replication stress and discovered ATR as a potential biomarker for WM-3835 treatment.

ATR↗
PubMed2004

ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation.

The BLM helicase, a deficiency that markedly increases cancer incidence in humans, is required for optimal repair during DNA replication. We show that BLM rapidly moves from PML nuclear bodies to damaged replication forks, returning to PML bodies several hours later, owing to activities of the DNA damage response kinases ATR and ATM, respectively. Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different sub-cellular compartments after replication stress. Unexpectedly, fibroblasts lacking BLM were deficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failed to form foci following replication stress. Expression of a dominant p53 mutant or helicase-deficient BLM restored replication stress-induced 53BP1 foci, but only mutant p53 restored optimal ATM activation. Thus, optimal repair of damaged replication fork lesions likely requires both ATR and ATM. BLM recruits 53BP1 to these lesions independent of its helicase activity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Adenosine Triphosphatases↗
PubMed2005

Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress.

The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. We have previously shown that destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation was not sufficient to explain the rapid destruction of Dup at G1-S. Here, we present evidence that it is DNA replication itself that triggers rapid Dup destruction. We find that a range of defects in DNA replication stabilize Dup protein and that this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress was cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, we find that cells with high levels of Dup also have elevated levels of Gem protein. We propose that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails.

Animals↗
PubMed2005

The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability.

Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response.

Aphidicolin↗
PubMed2026

A system-level metastable model of cancer evolution: integrating replication stress, cell cycle deregulation and chromosomal instability.

INTRODUCTION: Cancer cell proliferation occurs within the context of persistent genomic instability. In this review, we propose the RS-CCD-CIN axis as a systems-level framework in which replication stress (RS), cell cycle deregulation (CCD) and chromosomal instability (CIN) form an interdependent triad that shapes tumour evolution. This axis represents a constrained metastable state in which genomic instability is tolerated and buffered. The objective of this review is to synthesize the current understanding of how the RS-CCD-CIN axis contributes to tumour heterogeneity, adaptability and therapy response. DISCUSSION: Evidence indicates that RS, CCD and CIN operate as a dynamic, interconnected network rather than as independent processes. Replication stress induces DNA damage and mutagenesis, while partial checkpoint disruption permits cells with unresolved lesions to proliferate. Chromosomal instability generates both structural and numerical alterations, contributing to intratumoural heterogeneity. Together, these processes facilitate adaptation to environmental and therapeutic pressures. Extrachromosomal DNA, micronuclei formation and cytosolic DNA signalling, including the cGAS-STING pathway, connect genomic instability to adaptive responses and immune modulation. Single-cell and spatial profiling reveal temporal and spatial variability in RS, CCD and CIN states, highlighting the limitations of static biomarkers. Therapeutically, targeting individual components often yields limited durability, whereas approaches that simultaneously perturb multiple aspects of the RS-CCD-CIN axis may improve clinical outcomes. CONCLUSIONS: This review highlights the RS-CCD-CIN axis as a fragile and metastable architecture that supports cancer evolution, while also being susceptible to collapse. A deeper understanding of this interconnected framework may inform the development of therapeutic strategies and enhance the management of resistance.

Humans↗
PubMed2001

Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90- hematopoietic progenitors.

To further characterize hematopoietic "replicative stress" induced by bone marrow transplantation (BMT), the cell-cycle status of CD90+/- subsets of marrow CD34+ cells obtained 2 to 6 months after transplantation from 11 fully chimeric recipients was examined. Cycling profiles, derived by flow cytometry after staining with Hoechst 33342 and pyronin Y, were compared with those of 14 healthy marrow donors. Primitive CD34+CD90+ cells represented a smaller proportion of CD34+ cells in recipients (10% +/- 4% versus 19.6% +/- 5.3% in donors; P <.0001) and were more mitotically active, with the proportion of cells in S/G2/M nearly 4-fold higher than in donors (15.6% +/- 3% and 4.4% +/- 1.6%, respectively; P <.0001). By comparison, there was a modest increase in the proportion of CD34+CD90- progenitors in S/G2/M after BMT (10.9% +/- 1% vs 9.6% +/- 2% in donors; P =.04). Replicative stress after BMT is borne predominantly by cells in a diminished CD34+CD90+ population.

Adult↗
PubMed2025

Compensatory evolution to DNA replication stress is robust to nutrient availability.

Evolutionary repair refers to the compensatory evolution that follows perturbations in cellular processes. While evolutionary trajectories are often reproducible, other studies suggest they are shaped by genotype-by-environment (GxE) interactions. Here, we test the predictability of evolutionary repair in response to DNA replication stress-a severe perturbation impairing the conserved mechanisms of DNA synthesis, resulting in genetic instability. We conducted high-throughput experimental evolution on Saccharomyces cerevisiae experiencing constitutive replication stress, grown under different glucose availability. We found that glucose levels impact the physiology and adaptation rate of replication stress mutants. However, the genetics of adaptation show remarkable robustness across environments. Recurrent mutations collectively recapitulated the fitness of evolved lines and are advantageous across macronutrient availability. We also identified a novel role of the mediator complex of RNA polymerase II in adaptation to replicative stress. Our results highlight the robustness and predictability of evolutionary repair mechanisms to DNA replication stress and provide new insights into the evolutionary aspects of genome stability, with potential implications for understanding cancer development.

DNA Replication↗
PubMed2005

Frag1, a homolog of alternative replication factor C subunits, links replication stress surveillance with apoptosis.

We report the identification and characterization of a potent regulator of genomic integrity, mouse and human FRAG1 gene, a conserved homolog of replication factor C large subunit that is homologous to the alternative replication factor C subunits Elg1, Ctf18/Chl12, and Rad24 of budding yeast. FRAG1 was identified in a search for key caretaker genes involved in the regulation of genomic stability under conditions of replicative stress. In response to stress, Atr participates in the down-regulation of FRAG1 expression, leading to the induction of apoptosis through the release of Rad9 from damaged chromatin during the S phase of the cell cycle, allowing Rad9-Bcl2 association and induction of proapoptotic Bax protein. We propose that the Frag1 signal pathway, by linking replication stress surveillance with apoptosis induction, plays a central role in determining whether DNA damage is compatible with cell survival or whether it requires cell elimination by apoptosis.

Amino Acid Sequence↗
PubMed2005

Distinct modes of ATR activation after replication stress and DNA double-strand breaks in Caenorhabditis elegans.

ATM and ATR are key components of the DNA damage checkpoint. ATR primarily responds to UV damage and replication stress, yet may also function with ATM in the checkpoint response to DNA double-strand breaks (DSBs), although this is less clear. Here, we show that atl-1 (Caenorhabditis elegans ATR) and rad-5/clk-2 prevent mitotic catastrophe, function in the S-phase checkpoint and also cooperate with atm-1 in the checkpoint response to DSBs after ionizing radiation (IR) to induce cell cycle arrest or apoptosis via the cep-1(p53)/egl-1 pathway. ATL-1 is recruited to stalled replication forks by RPA-1 and functions upstream of rad-5/clk-2 in the S-phase checkpoint. In contrast, mre-11 and atm-1 are dispensable for ATL-1 recruitment to stalled replication forks. However, mre-11 is required for RPA-1 association and ATL-1 recruitment to DSBs. Thus, DNA processing controlled by mre-11 is important for ATL-1 activation at DSBs but not following replication fork stalling. We propose that atl-1 and rad-5/clk-2 respond to single-stranded DNA generated by replication stress and function with atm-1 following DSB resection.

Animals↗
PubMed2006

C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair.

One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.

Amino Acid Sequence↗
PubMed2005

Aspergillus nidulans uvsBATR and scaANBS1 genes show genetic interactions during recovery from replication stress and DNA damage.

The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsB(ATR) and the Mre11 complex (scaA(NBS1)). We demonstrate that there is an epistatic relationship between uvsB(ATR), the homolog of the ATR/MEC1 gene, and scaA(NBS1), the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaA(NBS1) expression during recovery from replication stress depends on uvsB(ATR). In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsB(ATR) and scaA(NBS1) function. Furthermore, ScaA(NBS1) is also dependent on uvsB(ATR) for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage.

Aspergillus nidulans↗
PubMed2006

Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis.

Cells of Bacillus subtilis actively co-ordinate the initiation of sporulation with DNA replication and repair. Conditions that perturb replication initiation or replication elongation induce expression of a small protein, Sda, that specifically inhibits the histidine kinases required to initiate spore development. Previously, the role of Sda has been studied during chronic blocks to DNA replication. Here we show that induction of Sda is required to delay the initiation of sporulation when replication elongation is transiently blocked or after UV irradiation. During the recovery phase, cells efficiently sporulated, but this required the proteolysis of Sda. The rapid proteolysis of Sda required the ClpXP protease and the uncharged C-terminal sequence of Sda. Replacing the last two residues of Sda, both serines, with aspartic acids markedly stabilized Sda. Strains expressing sdaDD from the endogenous sda locus were unable to efficiently initiate sporulation after transient replication stress. We conclude that the Sda replication checkpoint is required to delay the initiation of sporulation when DNA replication is transiently perturbed, and that the intrinsic instability of Sda contributes to shutting off the pathway. The Sda checkpoint thus co-ordinates early events of spore development, including the polar cell division, with successful completion of chromosome replication.

Amino Acid Sequence↗
PubMed2004

Domain organization of the yeast histone chaperone FACT: the conserved N-terminal domain of FACT subunit Spt16 mediates recovery from replication stress.

The abundant nuclear complex termed FACT affects several DNA transactions in a chromatin context, including transcription, replication, and repair. Earlier studies of yeast FACT, which indicated the apparent dispensability of conserved sequences at the N terminus of the FACT subunit Cdc68/Spt16, prompted genetic and biochemical studies reported here that suggest the domain organization for Spt16 and the other FACT subunit Pob3, the yeast homolog of the metazoan SSRP1 protein. Our findings suggest that each FACT subunit is a multidomain protein, and that FACT integrity depends on Pob3 interactions with the Spt16 Mid domain. The conserved Spt16 N-terminal domain (NTD) is shown to be without essential function during normal growth, but becomes important under conditions of replication stress. Genetic interactions suggest that some functions carried out by the Spt16 NTD may be partially redundant within FACT.

Amino Acid Sequence↗
PubMedDate not supplied

Claspin, a regulator of Chk1 in DNA replication stress pathway.

Regulation of the vertebrate checkpoint kinase Chk1 involves several protein complexes including the recently identified protein Claspin. Claspin associates with Chk1 upon replication stress and DNA damage and is required for Chk1 activation in both Xenopus and human systems. More importantly, Claspin is involved in regulation of cell cycle checkpoints. Here, we discuss the emerging roles of Claspin in the Chk1 pathway and its functions in checkpoint control.

Adaptor Proteins, Signal Transducing↗